match-standards-serum-mix08.Rmd

---
title: "Identifying mix08 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 <- 8
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.

### Aspirin

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

```{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, "WP", 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 mix08-serum-pos-aspirin-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- EICs show low intensity signal.

### Biotin

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

```{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, "WP", 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 and spectra with less than
2 peaks were removed).

```{r mix08-serum-pos-biotin-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 mix08-serum-pos-biotin-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 FT03054, FT02770 match with good similarity some of the
HMDB reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score  greater than 0.9).

```{r mix08-serum-pos-biotin-mirror-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)
```

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

```{r mix08-serum-pos-biotin-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)
```

```{r mix08-serum-pos-biotin-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_mbank, 0.9,
                           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

- FT03054 (RT = 72.01, ion `[M+H]+`) with confidence **A**.
- FT02770 (RT = 72.08, ion `[M+H-H2O]+`) with confidence **A**.
- FT03587 (RT = 72.13, ion `[M+Na]+`) with confidence **A-**.
- Reference MS2: FT03054 `[M+H]+`: FT03054_F07.S0259, FT03054_F08.S0287 (high
  confidence). FT02770 `[M+H-H2O]+`: FT02770_F08.S0264, FT02770_F08.S0310 (high
  confidence). FT03587 `[M+Na]+`: ; FT03587_F08.S0253 (low confidence).

### C5 Carnitine

```{r, echo = FALSE}
std <- "C5 Carnitine"
```

```{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, "WP", 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 mix08-serum-pos-c5-carnitine-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 mix08-serum-pos-c5-carnitine-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 mix08-serum-pos-c5-carnitine-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

- No spectra match.
- Maybe FT03089/FT03091 with confidence **D**??

### Cystathionine

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

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

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "WP", 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 mix08-serum-pos-cystathionine-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 mix08-serum-pos-cystathionine-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 FT02666, FT01015, FT02667 match with good similarity some
of the HMDB reference spectra for `r std`. Below we show the mirror plots of
the best matches (similarity score  greater than 0.92).

```{r mix08-serum-pos-cystathionine-mirror-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.92,
                           ppm = csp@ppm, tolerance = csp@tolerance)
```

```{r mix08-serum-pos-cystathionine-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 mix08-serum-pos-cystathionine-mirror-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.8,
                           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

- FT02666 (RT = 207.84, ion `[M+H]+`) with confidence **A**.
- FT02381 (RT = 207.84, ion `[M+H-H2O]+`) with confidence **A-**. But EIC
  doesn't appear very nice.
- FT03046 (RT = 208.12, ion `[M+Na]+`) with confidence **C**?
- FT01015 (RT = 208.51, ion `[M+2Na]2+`) with confidence **B**.
- FT02667 (RT = 222.21, ion `[M+H]+`) with confidence **A**?
- Reference MS2: FT02666 `[M+H]+`: FT02666_F07.S0730, FT02666_F07.S0760,
  FT02666_F07.S0793, FT02666_F08.S0770, FT02666_F08.S0838 (high confidence).
  FT03046 `[M+Na]+`: ; FT03046_F07.S0736, FT03046_F08.S0773 (low confidence)?
  FT01015 `[M+2Na]2+`: FT01015_F07.S0733, FT01015_F08.S0768 (high confidence).
  FT02667 `[M+H]+`: FT02667_F07.S0760, FT02667_F07.S0793,
  FT02667_F08.S0838 (high confidence)?


### 5,6-Dihydro Thymine

```{r, echo = FALSE}
std <- "5,6-Dihydro Thymine"
```

```{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, "WP", 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 mix08-serum-pos-5,6-dihydro-thymine-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 mix08-serum-pos-5,6-dihydro-thymine-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 mix08-serum-pos-5,6-dihydro-thymine-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

- No spectra match.
- No very convincing EIC.

### dGDP

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

```{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, "WP", 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 mix08-serum-pos-dgdp-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- EICs don't look very good.
  
### Glyceric Acid

```{r, echo = FALSE}
std <- "Glyceric 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 EIC for the assigned feature and visually inspect these.

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "WP", 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 mix08-serum-pos-glyceric-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 but we don't find any match.

```{r mix08-serum-pos-glyceric-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)
```

```{r mix08-serum-pos-glyceric-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, since the extracted spectra is associated to an ion
different from `[M+H]+` we perform a neutral loss spectra comparison but still
no match is found.

```{r mix08-serum-pos-glyceric-acid-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 mix08-serum-pos-glyceric-acid-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)
```
#### Summary

- No spectra match.

### Inosine

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

```{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, "WP", 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 mix08-serum-pos-inosine-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 mix08-serum-pos-inosine-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 mix08-serum-pos-inosine-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 ions different from
`[M+H]+` we perform a neutral loss spectra comparison and we find high
similarity matches mainly involving a single peak.

```{r mix08-serum-pos-inosine-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 mix08-serum-pos-inosine-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 mix08-serum-pos-norepinephrine-mbank_nl, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2_nl, std_mbank_nl, 0.9, 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)
```

For completeness, we also perform a neutral loss spectra comparison with HMDB
and MassBank.

```{r, echo = FALSE, message = FALSE, results = "asis", eval = ALL_NL_MATCH}
perform_match(std_ms2_nl, hmdb_nl, sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp_nl)
```

```{r, echo = FALSE, message = FALSE, results = "asis", eval = ALL_NL_MATCH}
perform_match(std_ms2_nl, mbank_nl,
              sv = c("rtime", "target_name", "score"),
              name = "target_name", param = csp_nl)
```

#### Summary

- FT05700 (RT = 144.89, ion `[M+2K-H]+`) with confidence **B**?
- FT04636 (RT = 145.44, ion `[M+K]+`) with confidence **B**?
- FT03635 (RT = 146.32, ion `[M+H]+`) with confidence **B-**?
- FT04186 (RT = 146.74, ion `[M+Na]+`) with confidence **B**?
- Reference MS2: FT05700 `[M+2K-H]+`: FT05700_F08.S0484 (high confidence)?
  FT04636 `[M+K]+`: FT04636_F07.S0465, FT04636_F08.S0483 (high confidence)?
  FT04186 `[M+Na]+`: FT04186_F07.S0474 (high confidence)?


### Mevalonic Acid

```{r, echo = FALSE}
std <- "Mevalonic 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, "WP", std_ms2)
```

The 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 mix08-serum-pos-mevalonic-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 but we don't find any match.

```{r mix08-serum-pos-mevalonic-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)
```

```{r mix08-serum-pos-mevalonic-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, since the extracted spectra are associated to an ion different from
`[M+H]+` we perform a neutral loss spectra comparison but still no match is
found.

```{r mix08-serum-pos-mevalonic-acid-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 mix08-serum-pos-mevalonic-acid-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)
```

#### Summary

- No spectra match.

### NADPH

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

```{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, "WP", 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 mix08-serum-pos-nadph-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- Low intensity signal EIC.

### Norepinephrine

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

```{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, "WP", 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 mix08-serum-pos-norepinephrine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra match.
- No very convincing EIC.

### Ornithine

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

```{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, "WP", 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 mix08-serum-pos-ornithine-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 mix08-serum-pos-ornithine-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 FT00678, FT00680, FT00688, FT01001 match with good
similarity some of the HMDB reference spectra for `r std`. Below we show the
mirror plots of the best matches (similarity score  greater than 0.8).

```{r mix08-serum-pos-ornithine-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)
```

With the MassBank comparison we don't get any match.

```{r mix08-serum-pos-ornithine-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

- FT00678 (RT = 191.17, ion `[M+H-NH3]+`) with confidence **B**? But seem to
  match better Proline.
- FT01475 (RT = 190.88, ion `[M+Na]+`) with confidence **B-**?
- FT01001 (RT = 249.14, ion `[M+H]+`) with confidence **B**? Not very convincing
  EIC
- Reference MS2: FT00678 `[M+H-NH3]+`: FT00678_F07.S0638 (high confidence)?
  FT01001 `[M+H]+`: FT01001_F08.S0875 (high confidence)?



### Succinic Acid

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

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

Three 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, "WP", 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 mix08-serum-pos-succinic-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 but we don't find any match.

```{r mix08-serum-pos-succinic-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)
```

```{r mix08-serum-pos-succinic-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

- No spectra match.

### Tyrosine

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

```{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, "WP", 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 mix08-serum-pos-tyrosine-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 mix08-serum-pos-tyrosine-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 feature FT01977 match with good similarity some of the HMDB
reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score  greater than 0.7).

```{r mix08-serum-pos-tyrosine-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 with the MassBank comparison as we show below.

```{r mix08-serum-pos-tyrosine-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

- FT01977 (RT = 168.96, ion `[M+H]+`) with confidence **B**.
- FT02738 (RT = 168.34, ion `[M+2Na-H]+`) with confidence **B-**.
- FT01673 (RT = 168.86, ion `[M+H-NH3]+`) with confidence **B-**.
- Reference MS2: FT01977 `[M+H]+`: FT01977_F07.S0573, FT01977_F08.S0599 (high
  confidence).

## Standards without any matching feature

### Uracil

# 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

### Aspirin

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

```{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, "WN", std_ms2)
```

The 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 mix08-serum-neg-aspirin-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- EICs with low intensity signal.

### Biotin

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

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

```{r, echo = FALSE}
plot_eics(data, std, feature_table, "WN", 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 mix08-serum-neg-biotin-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

```{r mix08-serum-neg-biotin-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 FT1671, FT2362, FT1670 match some of the HMDB reference
spectra for `r std`. Below we show the mirror plots of the best matches
(similarity score  greater than 0.7).

```{r mix08-serum-neg-biotin-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 with the MassBank comparison as shown below.

```{r mix08-serum-neg-biotin-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 mix08-serum-neg-biotin-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

- FT1671 (RT = 37.5, ion `[M-H]-`) with confidence **A**.
- FT2363 (RT = 37.94, ion `[M+HCOO]-`) with confidence **A-**.
- FT2183 (RT = 38.18, ion `[M+Cl]-`) with confidence **A-**.
- FT1670 (RT = 71.38, ion `[M-H]-`) with confidence **A**.
- FT2362 (RT = 71.14, ion `[M+HCOO]-`) with confidence **A-**.
- Reference MS2: FT1671 `[M-H]-`: FT1671_F15.S0144 (high confidence);
  FT1671_F16.S0150 (low confidence).
  FT1670 `[M-H]-`: FT1670_F16.S0246 (high confidence).
  FT2362 `[M+HCOO]-`: ; FT2362_F16.S0262 (low confidence).

### Cystathionine

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

```{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, "WN", 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 mix08-serum-neg-cystathionine-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 mix08-serum-neg-cystathionine-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 feature FT1369 match with good similarity some of the HMDB
reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score  greater than 0.7).

```{r mix08-serum-neg-cystathionine-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 find similar results for MassBank as shown below.

```{r mix08-serum-neg-cystathionine-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 mix08-serum-neg-cystathionine-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

- FT1862 (RT = 206.15, ion `[M+Cl]-`) with confidence **A-**.
- FT1369 (RT = 206.8, ion `[M-H]-`) with confidence **A**.
- Reference MS2: FT1369 `[M-H]-`: FT1369_F15.S0707, FT1369_F15.S0749,
  FT1369_F15.S0780, FT1369_F16.S0733 (high confidence).

### 5,6-Dihydro Thymine

```{r, echo = FALSE}
std <- "5,6-Dihydro Thymine"
```

```{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, "WN", 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 mix08-serum-neg-5,6-dihydro-thymine-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 mix08-serum-neg-5,6-dihydro-thymine-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 mix08-serum-neg-5,6-dihydro-thymine-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

- No spectra match.
- FT0218 (RT = 178.8, `[M-H]-` ion) with confidence **D**??

### Glyceric Acid

```{r, echo = FALSE}
std <- "Glyceric 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, "WN", 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 mix08-serum-neg-glyceric-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 mix08-serum-neg-glyceric-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)
```

Spectra from feature FT0110 match some of the HMDB reference spectra for
`r std`. Below we show the mirror plots of the best matches (similarity
score greater than 0.7)..

```{r mix08-serum-neg-glyceric-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 mix08-serum-neg-glyceric-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", "target_inchikey", "score"),
              name = "target_name", param = csp)
```

#### Summary

- FT0110 (RT = 75.82, ion `[M-H]-`) with confidence **A**.
- Reference MS2: FT0110 `[M-H]-`: FT0110_F15.S0292 (high confidence);
  FT0110_F15.S0245, FT0110_F16.S0254, FT0110_F16.S0306 (low confidence).


### Inosine

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

```{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, "WN", 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 mix08-serum-neg-inosine-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- FT1992 (RT = 145.6, ion `[M-H]-`) with confidence **D**?
- FT1992 (RT = 145.6, ion `[M-H]-`) with confidence **D**?

### Mevalonic Acid

```{r, echo = FALSE}
std <- "Mevalonic 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, "WN", 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 mix08-serum-neg-mevalonic-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 mix08-serum-neg-mevalonic-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)
```

```{r mix08-serum-neg-mevalonic-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)
```

#### Summary

- No spectra match.
- FT0310 (RT = 48.28, `[M-H]-` ion) with confidence **D**?
- FT2849 (RT = 47.51, `[M+HCOO]-` ion) with confidence **D**?

### NADPH

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

```{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, "WN", 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 mix08-serum-neg-nadph-ms2, echo = FALSE}
plot_spectra(std_ms2)
```

#### Summary

- No MS2 spectra available.
- Low intensity signal for the only available feature.

### Ornithine

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

```{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, "WN", std_ms2)
```

The 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 mix08-serum-neg-ornithine-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 good spectra match.

```{r mix08-serum-neg-ornithine-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 mix08-serum-neg-ornithine-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

- No spectra match.
- FT0277 (RT = 189.3, `[M-H]-` ion) with confidence **D**?

### Succinic Acid

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

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

Three 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, "WN", 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 mix08-serum-neg-succinic-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 mix08-serum-neg-succinic-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)
```

Spectra from feature FT0172 match with good similarity some of the HMDB
reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score  greater than 0.7).

```{r mix08-serum-neg-succinic-acid-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 mix08-serum-neg-succinic-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


- FT0172 (RT = 51.7, ion `[M-H]-`) with confidence **B**.
- Reference MS2: FT0172 `[M-H]-`: FT0172_F15.S0153, FT0172_F15.S0183,
  FT0172_F16.S0186 (high confidence).

### Tyrosine

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

```{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, "WN", 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 mix08-serum-neg-tyrosine-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 mix08-serum-neg-tyrosine-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 feature FT0794 match (with similarity lowe than 0.7)  some of the
HMDB reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score greater than 0.5).

```{r mix08-serum-neg-tyrosine-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 obtain similar results from the MassBank comparison as shown below.

```{r mix08-serum-neg-tyrosine-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
.
- FT0794 (RT = 168.04, ion `[M-H]-`) with confidence **C**.
- Reference MS2: FT0794 `[M-H]-`: FT0794_F15.S0559,
  FT0794_F15.S0619 (low confidence).

### Uracil

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

```{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, "WN", 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 mix08-serum-neg-uracil-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 mix08-serum-neg-uracil-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 feature FT0126 match with quite low similarity some of the HMDB
reference spectra for `r std`. Below we show the mirror plots of the best
matches (similarity score  greater than 0.7). For each of them only one peak
matches.

```{r mix08-serum-neg-uracil-hmdb, echo = TRUE, fig.cap = "Mirror plots"}
std_ms2_sel <- plot_select_ms2(std_ms2, std_hmdb, 0.3, ppm = csp@ppm,
                               tolerance = csp@tolerance)
```

We similar results with the MassBank comparison as shown below.

```{r mix08-serum-neg-uracil-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 mix08-serum-neg-uracil-mbank, echo = TRUE, fig.cap = "Mirror plots"}
tmp_sel <- plot_select_ms2(std_ms2, std_mbank, 0.3, 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


- FT0126 (RT = 65.69, ion `[M-H]-`) with confidence **C**?
- Reference MS2: FT0126 `[M-H]-`: ; FT0126_F16.S0191,
  FT0126_F16.S0235 (low confidence)?

## Standards without any matching feature

### C5 Carnitine

### dGDP

### Norepinephrine

# Summary on the ion database

# Session information

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

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