2.1 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(),
    ANNOTATE = TRUE
    )
} 

The library generation workflow was performed using the following parameters:

2.2 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_) )
}

2.3 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

2.4 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:  0.0109059731165568 minutes

2.5 Peptides used for RT normalization

The following peptides are used for the RT normalization. The last column indicates by FALSE/TRUE if a peptides is included in the data. The rows were ordered by the RT values.

3.1 Library Generation Summary

Total Number of PSM’s with Mascot e score < 0.05, in your search is 184. The number of unique precurosors is 137. The size of the generated ion library is 131. That means that 95.62 % of the unique precursors fullfilled 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:
##   ethods\20140910_01_fetuin_400amol_1.raw GAGSSEPVTGLDAK 
##       ethods\20140910_01_fetuin_400amol_1.raw TPVITGAPYEYR 
##       ethods\20140910_01_fetuin_400amol_1.raw VEATFGVDESNAK 
##       ethods\20140910_07_fetuin_400amol_2.raw ADVTPADFSEWSK 
##       ethods\20140910_07_fetuin_400amol_2.raw DGLDAASYYAPVR 
##       ethods\20140910_07_fetuin_400amol_2.raw GTFIIDPGGVIR 
##       ethods\20140910_07_fetuin_400amol_2.raw LFLQFGAQGSPFLK 
##       ethods\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 =    763000 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 
## rachse_20140910_Nuclei_diff_extraction_methods\20140910_07_fetuin_400amol_2.raw
## score = 15.83609 
## 
## size:
## Memory usage: 4224 bytes

plot(specLibrary@ionlibrary[[1]])

plot 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 .

plot(specLibrary)