match-standards-serum-mix06.Rmd

---
title: "Identifying mix06 standards in serum"
author: "Andrea Vicini, Vinicius Verri Hernandes, Johannes Rainer"
output:
  rmarkdown::html_document:
    highlight: pygments
    toc: true
    toc_float: true
    toc_depth: 3
    fig_width: 5
---

```{r, include = FALSE, cached = FALSE}
knitr::read_chunk("R/match-standards-chunks.R")
MIX <- 6
MATRIX <- "Serum"
POLARITY <- "POS"
## settings to find MS2 spectra for features. Be rather inclusive here.
FEATURE_MS2_PPM <- 20
FEATURE_MS2_TOLERANCE <- 0.05
```

```{r, libraries, echo = FALSE, message = FALSE}
```
```{r, general-settings, echo = FALSE, message = FALSE}
```

**Current version: 0.10.0, 2022-05-30.**

# Introduction

Mixes of standards have been solved in serum or added to human serum sample
pools in two different concentration and these samples were measured with the
LC-MS setup from Eurac (used also to generate the CHRIS untargeted metabolomics
data). Assignment of retention time and observed ion can be performed for each
standard based on MS1 information such as expected m/z but also based on the
expected difference in signal between samples with low and high concentration of
the standards. Finally, experimental fragment spectra provide the third level of
evidence. Thus, the present data set allows to annotate features to standards
based on 3 levels of evidence: expected m/z, difference in measured intensity
and MS2-based annotation.

A detailed description of the approach is given in
[match-standards-introduction.Rmd](match-standards-introduction.Rmd).


The list of standards that constitute the present sample mix are listed below
along with the expected retention time and the most abundant adduct for positive
and negative polarity as defined manually in a previous analysis by Mar
Garcia-Aloy.

```{r, standards-table, echo = FALSE, results = "asis"}
```

# Data import

We next load the data and split it according to matrix and polarity.

```{r, data-import}
```

We next also load the reference databases we will use to compare the
experimental MS2 spectra against. Below we thus load HMDB and MassBank data. We
create in addition *neutral loss* versions of the databases. Since HMDB does not
provide precursor m/z values we assume the fragment spectra to represent `[M+H]+`
ions (respectively `[M-H]-` ions for negative polarity) and add the m/z for
these adducts as *precursor m/z*.

```{r, load-reference-databases, message = FALSE}
```

```{r, setup-ion-db, message = FALSE, echo = FALSE}
```

# Initial evaluation of available MS2 data

Before performing the actual analysis that involves chromatographic peak
detection and evaluation of feature abundances, we compare all experimental MS2
spectra against the reference libraries. This provides already a first hint for
which standards a valid MS2 spectrum was recorded (and hence would allow
annotation based on evidence 3) and what related retention time might be.

We first extract all MS2 spectra from the respective data files and process them
by first removing all peaks with an intensity below 5% of the highest peak
signal, then scaling all intensities to a value range between 0 and 100 and
finally remove all MS2 spectra with less than 2 peaks.

```{r prepare-all-ms2}
fls <- fileNames(data_all)[data_all$polarity == "POS"]
```
```{r, all-ms2}
```

We next match these spectra against all reference spectra from HMDB for the
standards in `r MIX_NAME`. 

The settings for the matching are defined below. These will be used for all MS2
spectra similarity calculations. All matching spectra with a similarity larger
0.7 are identified.

```{r, compare-spectra-param}
```
 
Standards for which no MS2 spectrum in HMDB is present are listed in
the table below.

```{r, echo = FALSE, results = "asis"}
tmp <- std_dilution[!(std_dilution$HMDB %in% hmdb_std$compound_id), ]
pandoc.table(tmp[, c("name", "HMDB", "formula")], style = "rmarkdown",
             caption = "Standards for which no reference spectrum is available")
```

The table below lists all standards and the number of matching reference spectra
(along with their retention times).

```{r, table-all-ms2-hmdb, echo = FALSE, results = "asis"}
```

We repeat the same analysis for the negative polarity data (code not shown
again).

```{r, echo = FALSE}
fls <- fileNames(data_all)[data_all$polarity == "NEG"]
```
```{r, all-ms2, echo = FALSE}
```
```{r, table-all-ms2-hmdb, echo = FALSE, results = "asis"}
```

We next perform the full analysis of the data set involving MS1 and MS2 data.


# Serum, positive polarity

We first perform the analysis on the samples with the standards solved in pure
serum acquired in positive polarity mode.

```{r}
POLARITY <- "POS"
data <- filterFile(data_all, which(data_all$polarity == POLARITY))
hmdb <- hmdb_pos
hmdb_nl <- hmdb_pos_nl
```

## Data pre-processing

We perform the pre-processing of our data set which consists of the
chromatographic peak detection followed by a peak refinement step to reduce peak
detection artifacts, the correspondence analysis to group peaks across samples
and finally the gap-filling to fill in missing peak data from samples in which
no chromatographic peak was detected. For details on settings please refer to
the analysis of *mix 01* samples.

```{r, preprocessing, eval = !file.exists(paste0(RDATA_PATH, "processed_data_", POLARITY, ".RData")), message = FALSE}
```

```{r, echo = FALSE}
load(paste0(RDATA_PATH, paste0("processed_data_", POLARITY, ".RData")))
data_FS <- filterFile(data, which(data$mode == "FS"),
                      keepFeatures = TRUE)
```

## Signal intensity difference

We compute the difference in (log2) signals between samples with high and low
concentration of the standards and calculate the p-value for this difference
using the Student's t-test.

```{r, abundance-difference}
```

## Identification of features matching standards

We now identify all features matching the any of the pre-defined set of adducts
for the standards of mix `r MIX`. Matching features are further subsetted to
those that show an at least twice as high signal in samples with high
concentration compared to those with low concentrations. Also, features with a
signal in low concentration but no detectable signal in high concentration are
removed.

```{r, define-adducts-pos}
```
```{r, match-features}
```

For most of the standards (`r length(unique(mD$target_name))` out of 
`r nrow(std_dilution)`) in mix `r MIX` at least one feature was found
matching the standards adducts' m/z. 

```{r, table-standard-no-feature, results = "asis", echo = FALSE}
```

In the next sections we investigate for each standard which assignment would be
the correct one or, for those for which no signal was detected, why that was the
case.

## Standards with matching features

While for some standards a matching feature was found we still need to evaluate
whether this matching is correct. For each standard we thus first evaluate the
EICs for all matching features, then we match their MS2 spectra (if available)
against reference libraries. To ensure correct assignment of a feature, its
retention time and eventually related MS2 spectra, we consider the following
criteria to determine the annotation confidence:

- feature(s) was/were found with m/z matching those of adduct(s) of the 
  standard.
- signal is higher for samples with higher concentration.
- MS2 spectra matches reference spectra for the standard (if available).
- MS2 spectra don't match MS2 spectra of other compounds.

### Acetylglutamic acid

```{r, echo = FALSE}
std <- "Acetylglutamic acid"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EICs for the assigned features and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-acetylglutamic-acid-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-acetylglutamic-acid-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

With the HMDB comparison we find matches for spectra from features FT01829,
FT02249, FT02250, FT01830. The matches with similarity score greater than 0.7
are shown below.

```{r mix06-serum-pos-acetylglutamic-acid-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7,
                           ppm = csp@ppm, tolerance = csp@tolerance)
```

With the MassBank comparison we don't find any matches as we show below.

```{r mix06-serum-pos-acetylglutamic-acid-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- EIC is broad and noisy.
- FT02249 (RT=91.5, `[M+H]+` ion) with confidence level **A**.
- FT01829 (RT=91.2, `[M+H-H2O]` ion) with confidence level **A**.
- Reference MS2: `[M+H+]`: FT02249_F07.S0310, FT02249_F08.S0284; high
  confidence. `[M+H-H2O]`: FT01829_F07.S0309, FT01829_F08.S0283; high
  confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT02249_F07.S0310", "FT02249_F08.S0284", "FT01829_F07.S0309",
                 "FT01829_F08.S0283")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Acetylglycine

```{r, echo = FALSE}
std <- "Acetylglycine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned features and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The only available MS2 spectrum (cleaned) for the features matched to `r std`
is shown below (peaks with an intensity below 5% of the maximum peak and
spectra with less than 2 peaks were removed).

```{r mix06-serum-pos-acetylglycine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectrum against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-acetylglycine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

The extracted spectrum matches with similarity score lower than 0.7. The best
matches (shown below) involve only one peak.

```{r mix06-serum-pos-acetylglycine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.4,
                           ppm = csp@ppm, tolerance = csp@tolerance)
```

No match is found with MassBank comparison as shown below.

```{r mix06-serum-pos-acetylglycine-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```


```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT00777 (RT = 57.91, `[M+H]+` ion) with confidence level **C**.
- Reference MS2: `[M+H]+`: FT00777_F07.S0245, FT00777_F08.S0227; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT00777_F07.S0245", "FT00777_F08.S0227")]
ms2$confidence <- c("low")
```

```{r, add-ms2-spectra, echo = FALSE}
```

  
### Arginine

```{r, echo = FALSE}
std <- "Arginine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-arginine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-arginine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

All extracted spectra except FT02355_F08.S0666 seem to match some of the HMDB
reference spectra from `r std` to certain extent. The best matches involve
FT01902_F07.S0809, FT01904_F08.S0809, FT01896_F08.S1021, FT01899_F08.S0770,
FT01899_F08.S0770.

```{r mix06-serum-pos-arginine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.85,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

We obtain similar results with the MassBank comparison as we show below.

```{r mix06-serum-pos-arginine-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Assigning grade *A* even if we can't distinguish between Arginine, DL-Arginine
  and L-Arginine.
- FT01895 (RT = 188.8, `[M+H]+` ion) with confidence **A**.
- FT02355 (RT = 188.4, `[M+Na]+` ion) with confidence **A-**.
- FT00285 (RT = 188.8, `[M+2H]2+` ion) with confidence **A-**.
- FT01564 (RT = 188.7, `[M+H-H2O]+` ion) with confidence **A-**.
- FT01580 (RT = 188.1, `[M+H-NH3]+` ion) with confidence **A-**.
- Reference MS2: `[M+H]+`: FT01895_F07.S0626, FT01895_F07.S0674,
  FT01895_F08.S0665; high confidence. `[M+Na]+`: FT02355_F08.S0666; low
  confidence. `[M+H-NH3]+`: FT01580_F07.S0655, FT01580_F08.S0658; low
  confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT01895_F07.S0626", "FT01895_F07.S0674", "FT01895_F08.S0665",
                 "FT02355_F08.S0666", "FT01580_F07.S0655", "FT01580_F08.S0658")]
ms2$confidence <- c(rep("high", 3), rep("low", 3))
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Asparagine

```{r, echo = FALSE}
std <- "Asparagine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The extracted MS2 spectra for the features matched to `r std` are shown
below (peaks with an intensity below 5% of the maximum peak where removed).

```{r mix06-serum-pos-asparagine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectrum against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-asparagine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-pos-asparagine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

```{r mix06-serum-pos-asparagine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

```{r mix06-serum-pos-asparagine-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.7,
                           ppm = csp@ppm, tolerance = csp@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Grade *A* because the `[M+H]+` MS2 spectra all match only the standard. MS2
  spectra for the `[M+H-NH3]+` ion match also Proline with high similarity, but
  the EIC of the feature is highly similar to the one of the `[M+H]+` ion and
  shows the expected increase in concentration.
- FT01057 (RT = 187, `[M+H]+` ion) with confidence **A**.
- FT00738 (RT = 185, `[M+H-NH3]+` ion) with confidence **A-**.
- Reference MS2: `[M+H]+`: FT01057_F07.S0647, FT01057_F08.S0657; high
  confidence. `[M+H-NH3]+`: FT00738_F07.S0646, FT00738_F08.S0653,
  FT00738_F08.S0654; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT01057_F07.S0647", "FT01057_F08.S0657", "FT00738_F07.S0646",
                 "FT00738_F08.S0653", "FT00738_F08.S0654")]
ms2$confidence <- c(rep("high", 2), rep("low", 3))
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Choline

```{r, echo = FALSE}
std <- "Choline"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-choline-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-choline-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-pos-choline-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.75, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-pos-choline-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

```{r mix06-serum-pos-choline-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.75, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

Are Choline and 2-Hydroxyethyl trimethylammonium the same?

- Choline shows a very broad EIC. We're selecting the first feature/peak as the
  representative one.
- FT00499 (RT = 36.92, ion `[M+H]+`): confidence **A**.
- FT01240 (RT = 36.56, ion `[M+K]+`): confidence **A-**.
- FT00272 (RT = 35.76, ion `[M+H-H2O]+`): confidence **A-**.
- Reference MS2: `[M+H]+`: FT00499_F07.S0149, FT00499_F08.S0146; high
  confidence.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT00499", "FT01240", "FT00272"),
                  confidence_level = c("A", "A-", "A-"))

## Get MS2 spectra:
ms2 <- std_ms2[c("FT00499_F07.S0149", "FT00499_F08.S0146")]
ms2$confidence <- c("high")
```

```{r, add-ions, echo = FALSE}
```

```{r, add-ms2-spectra, echo = FALSE}
```


### CMP

```{r, echo = FALSE}
std <- "CMP"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from two
different compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The only available (cleaned) MS2 spectrum for the features matched to
`r std` is shown below (peaks with an intensity below 5% of the maximum peak
and spectra with less than 2 peaks were removed).

```{r mix06-serum-pos-cmp-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank but we don't find matches. 

```{r mix06-serum-pos-cmp-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-pos-cmp-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition, since the extracted spectrum is associated to an ion
different from `[M+H]+` we perform a neutral loss spectra comparison.

```{r mix06-serum-pos-cmp-ms2-hmdb_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_hmdb_nl <- hmdb_nl[hmdb_nl$compound_id == hmdb_id]
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_hmdb_nl, csp_nl)
```

```{r mix06-serum-pos-cmp-mirror-hmdb_nl, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2_nl, std_hmdb_nl, 0.5, ppm = csp_nl@ppm,
                           tolerance = csp_nl@tolerance)
```

The matches are not that good.

```{r mix06-serum-pos-cmp-ms2-mbank_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank_nl <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey), nl = TRUE)
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_mbank_nl, csp_nl)
```

```{r mix06-serum-pos-cmp-mirror-mbank_nl, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2_nl, std_mbank_nl, 0.5, ppm = csp_nl@ppm,
                           tolerance = csp_nl@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from negative polarity or water data. EIC data
  looks very good, but no evidence from MS2 spectra available.
- FT04915 (RT=212.6, `[M+H]+` ion) confidence level **D**.
- FT05366 (RT=212.9, `[M+Na]+` ion) confidence level **D**.
- FT05676 (RT=212.6, `[M+K]+` ion) confidence level **D**.
- Reference MS2: `[M+Na]+`: FT05366_F08.S0781; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT05366_F08.S0781")]
ms2$confidence <- c("low")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Cystine

```{r, echo = FALSE}
std <- "Cystine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-cystine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-cystine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

Spectra from features FT03052 and FT03054 match some of the HMDB reference
spectra for `r std`. The best matches (similarity score greater than 0.7) are
shown in the mirror plots below.
 
```{r mix06-serum-pos-cystine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

With the MassBank comparison we obtain similar results but with lower scores as
shown below.

```{r mix06-serum-pos-cystine-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "target_inchikey", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank,
              sv = c("rtime", "target_name", "target_inchikey", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Assigning grade *A* although we can not distinguish between Cystine and
  L-Cystine.
- FT03052 (RT = 213.6, `[M+H]+`): confidence level **A**.
- FT00826 (RT = 213.6, `[M+2H]2+`): confidence level **A-**.
- FT03389 (RT = 213.9, `[M+Na]+`): confidence level **A-**.
- Reference MS2: `[M+H]+`: FT03052_F07.S0761, FT03052_F07.S0781,
  FT03052_F07.S0804, FT03052_F08.S0801; high confidence. FT03052_F08.S0771; low
  confidence.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT03052", "FT00826", "FT03389"),
                  confidence_level = c("A", "A-", "A-"))

## Get MS2 spectra:
ms2 <- std_ms2[c("FT03052_F07.S0761", "FT03052_F07.S0781", "FT03052_F07.S0804",
                 "FT03052_F08.S0801", "FT03052_F08.S0771")]
ms2$confidence <- c(rep("high", 4), rep("low", 1))
```

```{r, add-ions, echo = FALSE}
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Histamine

```{r, echo = FALSE}
std <- "Histamine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-histamine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-histamine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```
 
```{r mix06-serum-pos-histamine-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.75, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-pos-histamine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- MS2 spectra of the `[M+Na]+` ion match also other compounds, but the EIC is
  very similar to the one of the `[M+H]+` ion and shows also the expected
  differences in concentrations.
- FT00652 (RT = 174.5, ion `[M+H]+`): confidence level **A**.
- FT01081 (RT = 172.7, ion `[M+Na]+`): confidence level **A-**.
- FT00380 (RT = 174.6, ion `[M+H-NH3]+`): confidence level **A-**.
- Reference MS2: `[M+H]+`: FT00652_F07.S0575, FT00652_F08.S0606; high
  confidence. `[M+Na]+`: FT01081_F07.S0577, FT01081_F07.S0578,
  FT01081_F08.S0565, FT01081_F08.S0566; low confidence. `[M+H-NH3]+`:
  FT00380_F07.S0587, FT00380_F08.S0564; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT00652_F07.S0575", "FT00652_F08.S0606", "FT01081_F07.S0577",
                 "FT01081_F07.S0578", "FT01081_F08.S0565", "FT01081_F08.S0566",
                 "FT00380_F07.S0587", "FT00380_F08.S0564")]
ms2$confidence <- c(rep("high", 2), rep("low", 6))
```

```{r, add-ms2-spectra, echo = FALSE}
```


### 3-Hydroxybutyric Acid

```{r, echo = FALSE}
std <- "3-Hydroxybutyric Acid"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect it.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-3-hydroxybutyric-acid-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- Inconclusive; needs validation from negative polarity or water data.


### Methioninesulfoxide

```{r, echo = FALSE}
std <- "Methioninesulfoxide"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-methioninesulfoxide-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-methioninesulfoxide-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

We obtain low similarity matches for spectra from features FT1327 and FT1329.

```{r mix06-serum-pos-methioninesulfoxide-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.1, ppm = csp@ppm,
                           tolerance = csp@tolerance)
```

Matches are OK-ish.

```{r mix06-serum-pos-methioninesulfoxide-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- While MS2 spectra similarity is low the EIC signal is pretty clear.
- FT01711 (RT = 185.1, `[M+H]+` ion) with confidence level **C**.
- FT02215 (RT = 183.2, `[M+Na]+` ion) with confidence level **C-**.
- FT01376 (RT = 185.6, `[M+H-H2O]+` ion) with confidence level **C-**.
- FT01401 (RT = 185, `[M+H-NH3]+` ion) with confidence level **C-**.
- Reference MS2: `[M+H]+`: FT01711_F07.S0635, FT01711_F08.S0639; low
  confidence. `[M+Na]+`: FT02215_F07.S0610; low confidence. `[M+H-NH3]+`:
  FT01401_F08.S0634, FT01401_F07.S0633; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT01711_F07.S0635", "FT01711_F08.S0639", "FT02215_F07.S0610",
                 "FT01401_F08.S0634", "FT01401_F07.S0633")]
ms2$confidence <- c("low")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Paracetamol

```{r, echo = FALSE}
std <- "Paracetamol"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-paracetamol-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

```{r mix06-serum-pos-paracetamol-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```


```{r mix06-serum-pos-paracetamol-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.9, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-pos-paracetamol-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT01460 (RT=40.07, `[M+H]+` ion) with confidence level **A**.
- Reference MS2: `[M+H]+`: FT01460_F07.S0174, FT01460_F08.S0171; high
  confidence.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT01460"),
                  confidence_level = c("A"))

## Get MS2 spectra:
ms2 <- std_ms2[c("FT01460_F07.S0174", "FT01460_F08.S0171")]
ms2$confidence <- c("high")
```

```{r, add-ions, echo = FALSE}
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Sphingosine-1-phosphate

```{r, echo = FALSE}
std <- "Sphingosine-1-phosphate"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-sphingosine-1-phosphate-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-sphingosine-1-phosphate-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

Spectra from features FT06037, FT06039, FT05686 match well HMDB reference
spectra for `r std`.

```{r mix06-serum-pos-sphingosine-1-phosphate-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.8, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-pos-sphingosine-1-phosphate-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT06037 (RT=146.7, `[M+H]+` ion) with confidence level **A**.
- FT06706 (RT=144.7, `[M+K]+` ion) with confidence level **A-**.
- FT07286 (RT=144.6, `[M+2K-H]+` ion) with confidence level **A-**.
- FT05686 (RT=148.3, `[M+H-H2O]+` ion) with confidence level **A**.
- Reference MS2: `[M+H]+`: FT06037_F07.S0425, FT06037_F07.S0481,
  FT06037_F08.S0411, FT06037_F08.S0470; high confidence. `[M+H-H2O]+`:
  FT05686_F07.S0424, FT05686_F07.S0480, FT05686_F08.S0410, FT05686_F08.S0469;
  high confidence. `[M+K]+`: FT06706_F08.S0412, FT06706_F07.S0426,
  FT06706_F07.S0485; low confidence. `[M+2K-H]+`: FT07286_F07.S0410,
  FT07286_F07.S0451, FT07286_F08.S0399, FT07286_F08.S0439; low confidence.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT07286"),
                  confidence_level = c("A-"))

## Get MS2 spectra:
ms2 <- std_ms2[c("FT06037_F07.S0425", "FT06037_F07.S0481", "FT06037_F08.S0411",
                 "FT06037_F08.S0470", "FT05686_F07.S0424", "FT05686_F07.S0480",
                 "FT05686_F08.S0410", "FT05686_F08.S0469", "FT06706_F08.S0412",
                 "FT06706_F07.S0426", "FT06706_F07.S0485", "FT07286_F07.S0410",
                 "FT07286_F07.S0451", "FT07286_F08.S0399", "FT07286_F08.S0439")]
ms2$confidence <- c(rep("high", 8), rep("low", 7))
```

```{r, add-ions, echo = FALSE}
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Sucrose

```{r, echo = FALSE}
std <- "Sucrose"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-sucrose-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-sucrose-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-pos-sucrose-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

No match is found against HMDB or MassBank reference spectra for `r std`. Since
the extracted spectra are associated to an ion different from `[M+H]+` we
perform a neutral loss spectra comparison but still we don't find any match as
shown below.

```{r mix06-serum-pos-sucrose-ms2-hmdb_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_hmdb_nl <- hmdb_nl[hmdb_nl$compound_id == hmdb_id]
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_hmdb_nl, csp_nl)
```

```{r mix06-serum-pos-sucrose-ms2-mbank_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank_nl <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey), nl = TRUE)
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_mbank_nl, csp_nl)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from negative polarity or water data.
- FT04949 (RT=173.1, `[M+H-H2O]+` ion) with confidence level **D**.


### Tryptophan

```{r, echo = FALSE}
std <- "Tryptophan"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The only available MS2 spectrum (cleaned) for the features matched to `r std` is
shown below (peaks with an intensity below 5% of the maximum peak and spectra
with less than 2 peaks were removed).

```{r mix06-serum-pos-tryptophan-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-pos-tryptophan-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

Spectra from features FT02489, FT02218 match with high similarity
scores. The best matches (similarity score greater than 0.9) are shown in
the mirror plots below.

```{r mix06-serum-pos-tryptophan-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.8, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

We obtain similar results with the MassBank comparison as we show below.

```{r mix06-serum-pos-tryptophan-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Assigning grade *A* although we can not distinguish Tryptophan, L-Tryptophan
  and D-Tryptophan.
- FT02489 (RT= 155.6, `[M+H]+` ion) with confidence level **A**.
- FT02855 (RT= 153.2, `[M+Na]+` ion) with confidence level **A-**.
- FT03188 (RT= 155.8, `[M+2Na-H]+` ion) with confidence level **A-**.
- FT02218 (RT= 155.6, `[M+H-NH3]+` ion) with confidence level **A**.
- FT03088 (RT = 149.4, `[M+K]+` ion) with confidence level **A-**.
- Reference MS2: `[M+H]+`: FT02489_F07.S0475, FT02489_F08.S0451; high
  confidence. `[M+H-NH3]+`: FT02218_F07.S0474, FT02218_F08.S0450; high
  confidence. `[M+Na]+`: FT02855_F07.S0476; low confidence. `[M+2Na-H]+`:
  FT03188_F08.S0452; low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT02489_F07.S0475", "FT02489_F08.S0451", "FT02218_F07.S0474",
                 "FT02218_F08.S0450", "FT02855_F07.S0476", "FT03188_F08.S0452")]
ms2$confidence <- c(rep("high", 4), rep("low", 2))
```

```{r, add-ms2-spectra, echo = FALSE}
```


### UDP-acetylglucosamine

```{r, echo = FALSE}
std <- "UDP-acetylglucosamine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SP", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-pos-udp-acetylglucosamine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank but we don't find any match.

```{r mix06-serum-pos-udp-acetylglucosamine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-pos-udp-acetylglucosamine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

We also perform neutral loss comparison for spectra FT5874_F08.S0878 because
the associated ion is different from `[M+H]+` but no good match is found.

```{r mix06-serum-neg-udp-acetylglucosamine-ms2-hmdb_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_hmdb_nl <- hmdb_nl[hmdb_nl$compound_id == hmdb_id]
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_hmdb_nl, csp_nl)
```

```{r mix06-serum-neg-udp-acetylglucosamine-ms2-mbank_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank_nl <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey), nl = TRUE)
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_mbank_nl, csp_nl)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from negative polarity or water data. No
  spectra matches are found. EIC data for the `[M+H-NH3]+` ion looks good.
- FT09113 (RT=157, `[M+H-NH3]+` ion) with confidence level **D**.
- Reference MS2: `[M+H-NH3]+`: FT09113_F07.S0528, FT09113_F08.S0506; low
  confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT09113_F07.S0528", "FT09113_F08.S0506")]
ms2$confidence <- c("low")
```

```{r, add-ms2-spectra, echo = FALSE}
```


# Serum, negative polarity

We now perform the analysis on the samples with the standards still solved in
pure serum but acquired in negative polarity mode.

```{r}
POLARITY <- "NEG"
data <- filterFile(data_all, which(data_all$polarity == POLARITY))
hmdb <- hmdb_neg
hmdb_nl <- hmdb_neg_nl
```

```{r, preprocessing, eval = !file.exists(paste0(RDATA_PATH, "processed_data_", POLARITY, ".RData")), message = FALSE}
```

```{r, echo = FALSE}
load(paste0(RDATA_PATH, paste0("processed_data_", POLARITY, ".RData")))
data_FS <- filterFile(data, which(data$mode == "FS"),
                      keepFeatures = TRUE)
```


## Signal intensity difference

We compute the difference in (log2) signals between samples with high and low
concentration of the standards and calculate the p-value for this difference
using the Student's t-test.

```{r, abundance-difference}
```

## Identification of features matching standards

We now identify all features matching the any of the pre-defined set of adducts
for the standards of mix `r MIX`. Matching features are further subsetted to
those that show an at least twice as high signal in samples with high
concentration compared to those with low concentrations. Also, features with a
signal in low concentration but no detectable signal in high concentration are
removed.

```{r, define-adducts-neg}
```
```{r, match-features}
```

For most of the standards (`r length(unique(mD$target_name))` out of 
`r nrow(std_dilution)`) in mix `r MIX` at least one feature was found
matching the standards adducts' m/z. 

```{r, table-standard-no-feature, results = "asis", echo = FALSE}
```

In the next sections we investigate for each standard which assignment would be
the correct one or, for those for which no signal was detected, why that was the
case.

## Standards with matching features

### Acetylglutamic acid

```{r, echo = FALSE}
std <- "Acetylglutamic acid"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EICs for the assigned features and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-acetylglutamic-acid-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-acetylglutamic-acid-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

MS2 spectra (all from features FT0818) seem to match the HMDB reference spectra
with good similarity scores.

```{r mix06-serum-neg-acetylglutamic-acid-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

```{r mix06-serum-neg-acetylglutamic-acid-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

No spectrum matches reference spectra from MassBank.

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT0818 (RT = 90.86, `[M-H]-` ion) with confidence level **A**.
- Reference MS2: `[M-H]-`: FT0818_F15.S0190, FT0818_F15.S0223, FT0818_F15.S0251,
  FT0818_F15.S0288, FT0818_F16.S0261, FT0818_F16.S0280; high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT0818_F15.S0190", "FT0818_F15.S0223", "FT0818_F15.S0251",
                 "FT0818_F15.S0288", "FT0818_F16.S0261", "FT0818_F16.S0280")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Acetylglycine

```{r, echo = FALSE}
std <- "Acetylglycine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

A single feature has been assigned to this standard based on
m/z matching. We next plot its EIC and visually inspect it (has very
low intensity).

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-acetylglycine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

```{r mix06-serum-neg-acetylglycine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-neg-acetylglycine-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-neg-acetylglycine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT0135 (RT = 56.04, `[M-H]-` ion) with confidence level **A**.
- FT0530 (RT = 55.78, `[M+HCOO]-` ion) with confidence level **A-**.
- Reference MS2: `[M-H]-`: FT0135_F15.S0195, FT0135_F15.S0229, FT0135_F16.S0187;
  high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT0135_F15.S0195", "FT0135_F15.S0229", "FT0135_F16.S0187")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Arginine

```{r, echo = FALSE}
std <- "Arginine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

A single feature has been assigned to this standard based on
m/z matching. We next plot its EIC and visually inspect it (has very
low intensity).

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-arginine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-arginine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-neg-arginine-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

```{r mix06-serum-neg-arginine-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT0657 (RT = 186.5 with, `[M-H]-` ion) with confidence level **A**.
- FT1213 (RT = 185.8 with, `[M+HCOO]-` ion) with confidence level **A-**.
- Reference MS2: `[M-H]-`: FT0657_F15.S0625; high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT0657_F15.S0625")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Asparagine

```{r, echo = FALSE}
std <- "Asparagine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-asparagine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-asparagine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

The available spectra match but with similarity score lower than 0.7. Those
with similarity score higher than 0.5 are shown in the mirror plots below.

```{r mix06-serum-neg-asparagine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.5,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

We find similar results for MassBank as shown below.

```{r mix06-serum-neg-asparagine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

```{r mix06-serum-neg-asparagine-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.5,
                           ppm = csp@ppm, tolerance = csp@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT0244 (RT = 184.4, `[M-H]-` ion) with confidence level **C**.
- Reference MS2: `[M-H-]`: FT0244_F15.S0623, FT0244_F15.S0624, FT0244_F16.S0610;
  low confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT0244_F15.S0623", "FT0244_F15.S0624", "FT0244_F16.S0610")]
ms2$confidence <- c("low")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### CMP

```{r, echo = FALSE}
std <- "CMP"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-cmp-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-cmp-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

All the extracted spectra match reference spectra from HMDB.

```{r mix06-serum-neg-cmp-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

We repeat the same with MassBank and we obtain similar results as shown below.

```{r mix06-serum-neg-cmp-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

```{r mix06-serum-neg-cmp-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.7, ppm = csp@ppm,
                           tolerance = csp@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT2674 (RT = 209.9, `[M-H]-` ion) with confidence level **A**.
- FT3453 (RT = 209.8, `[M+HCOO]-` ion) with confidence level **A-**.
- Reference MS2: `[M-H]-`: FT2674_F15.S0710, FT2674_F15.S0736, FT2674_F15.S0753,
  FT2674_F16.S0697, FT2674_F16.S0744; high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT2674_F15.S0710", "FT2674_F15.S0736", "FT2674_F15.S0753",
                 "FT2674_F16.S0697", "FT2674_F16.S0744")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Cystine

```{r, echo = FALSE}
std <- "Cystine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-cystine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-cystine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```
 
```{r mix06-serum-neg-cystine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.8,
                               ppm = csp@ppm, tolerance = csp@tolerance)
```

We repeat the same with MassBank but we don't obtain any good match.

```{r mix06-serum-neg-cystine-acid-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank,
              sv = c("rtime", "target_name", "target_inchikey", "score"),
              name = "target_name", param = csp)
```

We don't find any match exept low similarity ones for 

#### Summary

- FT1428 (RT = 210.4, `[M-H]-` ion) with confidence **A**.
- Reference MS2: `[M-H]-`: FT1428_F15.S0717, FT1428_F15.S0740, FT1428_F15.S0773,
  FT1428_F16.S0696, FT1428_F16.S0722; high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT1428_F15.S0717", "FT1428_F15.S0740", "FT1428_F15.S0773",
                 "FT1428_F16.S0696", "FT1428_F16.S0722")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Histamine

```{r, echo = FALSE}
std <- "Histamine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-histamine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-histamine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-neg-histamine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition, since the extracted spectra are associated to an ion different
from `[M-H]-` we perform a neutral loss spectra comparison.

```{r mix06-serum-neg-histamine-ms2-hmdb_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_hmdb_nl <- hmdb_nl[hmdb_nl$compound_id == hmdb_id]
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_hmdb_nl, csp_nl)
```

```{r mix06-serum-neg-histamine-ms2-mbank_nl, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank_nl <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey), nl = TRUE)
std_ms2_nl <- neutralLoss(std_ms2, nl_param)
sim <- plot_ms2_similarity_heatmap(std_ms2_nl, std_mbank_nl, csp_nl)
```

We don't find any match (exept very low similarity ones with MassBank neutral
loss comparison).

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from positive or water data. EIC signal is low.


### 3-Hydroxybutyric Acid

```{r, echo = FALSE}
std <- "3-Hydroxybutyric Acid"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-3-hydroxybutyric-acid-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- Inconclusive; needs validation from positive polarity and water data.
- FT0073 (RT = 38.2, `[M-H]-` ion) with confidence level **D**.
- FT0418 (RT = 36.4, `[M+HCOO]-` ion) with confidence level**D**.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT0073", "FT0418"),
                  confidence_level = c("D", "D"))
```

```{r, add-ions, echo = FALSE}
```


### Methioninesulfoxide

```{r, echo = FALSE}
std <- "Methioninesulfoxide"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-methioninesulfoxide-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-methioninesulfoxide-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-neg-methioninesulfoxide-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from positive polarity or water data. EIC data
  looks however good.
- FT0550 (RT=182.8, `[M-H]-` ion) with confidence level **D**.
- FT0974 (RT=180.7, `[M+Cl]-` ion) with confidence level **D**.
- FT1097 (RT=182.8, `[M+HCOO]-` ion) with confidence level **D**.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT0550", "FT0974", "FT1097"),
                  confidence_level = c("D", "D", "D"))
```

```{r, add-ions, echo = FALSE}
```


### Paracetamol


```{r, echo = FALSE}
std <- "Paracetamol"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-paracetamol-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- Inconclusive; needs validation from positive polarity and water data. EIC data
  looks however good.
- FT0422 (RT = 36.38, `[M-H]-` ion) with confidence **D**.
- FT0922 (RT = 36.28, `[M+HCOO]-` ion) with confidence **D**.


### Sphingosine-1-phosphate

```{r, echo = FALSE}
std <- "Sphingosine-1-phosphate"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

Many features have been assigned to this standard based on m/z matching. Based
on their retention times, these seem however to represent ions from different
compounds.

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-sphingosine-1-phosphate-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-sphingosine-1-phosphate-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

```{r mix06-serum-neg-sphingosine-1-phosphate-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; needs validation from positive polarity and water data. The
  features listed below look promising.
- FT3630 (RT = 145.2, `[M-H]-` ion) with confidence level **D**.
- FT4453 (RT = 144.1, `[M+HCOO]-` ion) with confidence level **D**.


### Sucrose

```{r, echo = FALSE}
std <- "Sucrose"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectrum for the feature matched to `r std` is shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-sucrose-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-sucrose-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

We find matches for all the extracted spectra.

```{r mix06-serum-neg-sucrose-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

We obtain similar results from the MassBank comparison as shown below.

```{r mix06-serum-neg-sucrose-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT3027 (RT = 186.6, `[M-H]-` ion) confidence level **B**.
- FT3809 (RT = 186.5, `[M+HCOO]-` ion) confidence level **B-**.
- MS2 spectra:`[M-H]-`: FT3027_F16.S0628; high confidence.

```{r, echo = FALSE}
## Get MS2 spectra:
ms2 <- std_ms2[c("FT3027_F16.S0628")]
ms2$confidence <- c("high")
```

```{r, add-ms2-spectra, echo = FALSE}
```


### Tryptophan

```{r, echo = FALSE}
std <- "Tryptophan"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The only available MS2 spectrum (cleaned) for the features matched to `r std` is
shown below (peaks with an intensity below 5% of the maximum peak and spectra
with less than 2 peaks were removed).

```{r mix06-serum-neg-tryptophan-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- Inconclusive; needs validation from positive polarity and water data. The EIC
  data is however nice.
- FT1015 (RT = 152.4, `[M-H]-` ion) with confidence level **D**.
- FT1544 (RT = 155, `[M+HCOO]-` ion) with confidence level **D**.
- FT1429 (RT = 155.5, `[M+Cl]-` ion) with confidence level **D**.


### UDP-acetylglucosamine

```{r, echo = FALSE}
std <- "UDP-acetylglucosamine"
```

```{r, table-feature-matches, results = "asis", echo = FALSE, message = FALSE}
```

We next plot the EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "SN", std_ms2)
```

The cleaned MS2 spectra for the features matched to `r std` are shown below
(peaks with an intensity below 5% of the maximum peak and spectra with less
than 2 peaks were removed).

```{r mix06-serum-neg-udp-acetylglucosamine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

We next match the extracted MS2 spectra against the reference spectra for 
`r std` from HMDB and MassBank.

```{r mix06-serum-neg-udp-acetylglucosamine-ms2-hmdb, echo = FALSE, fig.width = 5, fig.height = 5}
hmdb_id <- std_dilution$HMDB[std_dilution$name == std]
std_hmdb <- Spectra(cdb, filter = ~ compound_id == hmdb_id)
sim <- plot_ms2_similarity_heatmap(std_ms2, std_hmdb, csp)
```

We find matches for all the extracted spectra.

```{r mix06-serum-neg-udp-acetylglucosamine-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.7, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

We obtain matches also with the MassBank comparison but with lower similarity
scores.

```{r mix06-serum-neg-udp-acetylglucosamine-ms2-mbank, echo = FALSE, fig.width = 5, fig.height = 5}
std_mbank <- get_mbank(mbank, inchikey = unique(std_hmdb$inchikey))
sim <- plot_ms2_similarity_heatmap(std_ms2, std_mbank, csp)
```

```{r mix06-serum-neg-udp-acetylglucosamine-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.4, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

In addition we match (**all**) the MS2 spectra for the matched features against
all spectra from HMDB or MassBank identifying reference spectra with a
similarity larger than 0.7. The results (if any spectra matched) are shown in
the two following tables.

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, hmdb, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

```{r, echo = FALSE, message = FALSE, results = "asis"}
perform_match(std_ms2, mbank, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp)
```

#### Summary

- Inconclusive; there are several features with matching MS2 spectra along a
  large RT period. EIC data looks not too good.
- FT6831 (RT= 329.1, `[M-H]-` ion) with confidence level **A**.
- FT6834 (RT= 208.6, `[M-H]-` ion) with confidence level **A**.
- Reference MS2: `[M-H]-`: FT6831_F16.S0949, FT6834_F16.S0693; high confidence.

```{r, echo = FALSE}
## Define the feature (ion) and MS2 spectra to insert
fts <- data.frame(feature_id = c("FT6831"),
                  confidence_level = c("A"))

## Get MS2 spectra:
ms2 <- std_ms2[c("FT6831_F16.S0949", "FT6834_F16.S0693")]
ms2$confidence <- c("high")
```

```{r, add-ions, echo = FALSE}
```

```{r, add-ms2-spectra, echo = FALSE}
```


## Standards without any matching feature

### Choline


# Summary on the ion database

Summarizing the content that was added to the `IonDb`.

```{r, iondb-summary, echo = FALSE, results = "asis"}
```

# Session information

The R version and packages used in this analysis are listed below.

```{r sessioninfo}
sessionInfo()
```