Parameter

If no INPUT is defined, the report uses the specL package’s data and the following default parameters.

if(!exists("INPUT")){
  INPUT <- list(FASTA_FILE 
      = system.file("extdata", "SP201602-specL.fasta.gz",
                    package = "specL"),
    BLIB_FILTERED_FILE 
      = system.file("extdata", "peptideStd.sqlite",
                    package = "specL"),
    BLIB_REDUNDANT_FILE 
      = system.file("extdata", "peptideStd_redundant.sqlite",
                    package = "specL"),
    MIN_IONS = 5,
    MAX_IONS = 6,
    MZ_ERROR = 0.05,
    MASCOTSCORECUTOFF = 17,
    FRAGMENTIONMZRANGE = c(300, 1250),
    FRAGMENTIONRANGE = c(5, 200),
    NORMRTPEPTIDES = specL::iRTpeptides,
    OUTPUT_LIBRARY_FILE = tempfile(fileext ='.csv'),
    RDATA_LIBRARY_FILE = tempfile(fileext ='.RData'),
    ANNOTATE = TRUE
    )
} 

The library generation workflow was performed using the following parameters:

Define the fragment ions of interest

The following R helper function is used for composing the in-silico fragment ions using protViz.

fragmentIonFunction_specL <- function (b, y) {
  Hydrogen <- 1.007825
  Oxygen <- 15.994915
  Nitrogen <- 14.003074
  b1_ <- (b )
  y1_ <- (y )
  b2_ <- (b + Hydrogen) / 2
  y2_ <- (y + Hydrogen) / 2 
  return( cbind(b1_, y1_, b2_, y2_) )
}

Read the sqlite files

BLIB_FILTERED <- read.bibliospec(INPUT$BLIB_FILTERED_FILE) 

## fetched 137 rows.

## assigning 28 modifications ...

summary(BLIB_FILTERED)

## Summary of a "psmSet" object.
## Number of precursor:
##  137
## Number of precursors in Filename(s)
##  _methods\20140910_01_fetuin_400amol_1.raw   21
##  _methods\20140910_07_fetuin_400amol_2.raw   116
## Number of annotated precursor:
##  0

BLIB_REDUNDANT <- read.bibliospec(INPUT$BLIB_REDUNDANT_FILE) 

## fetched 184 rows.

## assigning 37 modifications ...

summary(BLIB_REDUNDANT)

## Summary of a "psmSet" object.
## Number of precursor:
##  184
## Number of precursors in Filename(s)
##  _methods\20140910_01_fetuin_400amol_1.raw   32
##  _methods\20140910_07_fetuin_400amol_2.raw   152
## Number of annotated precursor:
##  0

Protein (re)-annotation

After processing the psm using bibliospec, the protein information is gone. The read.fasta function is provided by the CRAN package seqinr.

if(INPUT$ANNOTATE){
  FASTA <- read.fasta(INPUT$FASTA_FILE, 
                    seqtype = "AA", 
                    as.string = TRUE)

  BLIB_FILTERED <- annotate.protein_id(BLIB_FILTERED, 
                                       fasta = FASTA)
}

## start protein annotation ...

## time taken:  0.000780916213989258 minutes

Peptides used for RT normalization

The following peptides are used for retention time (RT) normalization. The last column indicates by FALSE|TRUE if a peptide is included in the data. The rows were ordered by the RT values.

Library Generation Summary

Total Number of PSM’s with Mascot e-value < 0.05, in your search, is 184. The number of unique precursors is 137. The size of the generated ion library is 131. That means that 95.62 % of the unique precursors fulfilled the filtering criteria.

summary(specLibrary)

## Summary of a "specLSet" object.
## 
## Parameter:
## 
## Number of precursor (q1 and peptideModSeq) = 131
## Number of unique precursor
## (q1.in-silico and peptideModSeq) = 122
## Number of iRT peptide(s) = 8
## Which std peptides (iRTs) where found in which raw files:
##   _methods\20140910_01_fetuin_400amol_1.raw GAGSSEPVTGLDAK 
##       _methods\20140910_01_fetuin_400amol_1.raw TPVITGAPYEYR 
##       _methods\20140910_01_fetuin_400amol_1.raw VEATFGVDESNAK 
##       _methods\20140910_07_fetuin_400amol_2.raw ADVTPADFSEWSK 
##       _methods\20140910_07_fetuin_400amol_2.raw DGLDAASYYAPVR 
##       _methods\20140910_07_fetuin_400amol_2.raw GTFIIDPGGVIR 
##       _methods\20140910_07_fetuin_400amol_2.raw LFLQFGAQGSPFLK 
##       _methods\20140910_07_fetuin_400amol_2.raw TPVISGGPYEYR 
## 
## Number of transitions frequency:
##  5   16
##  6   115
## 
## Number of annotated precursor = 1855
## Number of file(s)
##  2
## 
## Number of precursors in Filename(s)
##  _methods\20140910_01_fetuin_400amol_1.raw   19
##  _methods\20140910_07_fetuin_400amol_2.raw   112
## 
## Misc:
## 
## Memory usage =    742776 bytes

In the following two code snippets the first element of the ion library is displayed:

#  slotNames(specLibrary@ionlibrary[[1]])
specLibrary@ionlibrary[[1]]

## An "specL" object.
## 
## 
## content:
## group_id = ADQPQC[+57.0]LSLAWSTDGQTLFAGYSDNTIR.3 
## peptide_sequence = ADQPQCLSLAWSTDGQTLFAGYSDNTIR 
## proteinInformation = sp|O18640|GBLP_DROME 
## q1 = 1039.151 
## q1.in_silico = 3172.464 
## q3 = 925.436 1143.542 996.4756 705.3505 868.4149 503.2933 
## q3.in_silico = 925.4374 1143.543 996.4745 705.3526 868.4159 503.2936 
## prec_z = 3 
## frg_type = y y y y y y 
## frg_nr = 8 10 9 6 7 4 
## frg_z = 1 1 1 1 1 1 
## relativeFragmentIntensity = 100 56 56 35 14 11 
## irt = 95.97 
## peptideModSeq = ADQPQC[+57.0]LSLAWSTDGQTLFAGYSDNTIR 
## mZ.error = 0.001407 0.001031 0.001095 0.002066 0.001004 0.000286 
## \ctrachse_20140910_Nuclei_diff_extraction_methods\20140910_07_fetuin_400amol_2.raw
## score = 15.83609 
## 
## size:
## Memory usage: 4224 bytes

plot(specLibrary@ionlibrary[[1]])

plot(specLibrary)

## [1] 16.83185 13.13262 18.54058 18.36923 15.30478 15.30478

## [1]  7.032372  6.490769 14.787681 14.544429 15.207398 15.207398

The code snippet below plots an overview of the whole ion library. Please note that the iRT peptides used for the normalization of RT do not have to be included in the resulting .