Accurate peptide fragment mass analysis: multiplexed peptide identification and quantification.

  • Belov M
  • Grinfeld D
  • Compton P
 et al. 
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The strategy of sequential window acquisition of all theoretical fragment ion spectra (SWATH) is emerging in the field of label-free proteomics. A critical consideration for the processing of SWATH data is the quality of the ion library (or mass spectrometric reference map). As the availability of open spectral libraries that can be used to process SWATH data is limited, most users currently create their libraries in-house. Herein, we propose an approach to construct an expanded ion library using the data-dependent acquisition (DDA) data generated by fractionation proteomics. We identified three critical elements for achieving a satisfactory ion library during the iterative process of our ion library expansion, including a correction of the retention times (RTs) gained from fractionation proteomics, appropriate integrations of the fractionated proteomics into an ion library, and assessments of the impact of the expanded ion libraries to data mining in SWATH. Using a bacterial lysate as an evaluation material, we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate the lysate proteins and constructed the expanded ion library using the fractionation proteomics data. Compared with the ion library built from the unfractionated proteomics, approximately 20% more peptides were extracted from the expanded ion library. The extracted peptides, moreover, were acceptable for further quantitative analysis.

Author-supplied keywords

  • Absolute quantitative analysis
  • Algorithms
  • Amino Acid Sequence
  • Automatic Data Processing
  • Automatic Data Processing: methods
  • Bacterial Proteins
  • Bacterial Proteins: analysis
  • Blood Proteins
  • Blood Proteins: analysis
  • Bovine
  • CID
  • Chromatography
  • Computational Biology
  • Data Independent Acquisition
  • Escherichia coli Proteins
  • Fourier Analysis
  • HCD
  • High Pressure Liquid
  • Humans
  • Ions
  • Ions: metabolism
  • Limit of Detection
  • Linear Models
  • Liquid
  • Mass Spectrometry
  • Mass Spectrometry: methods
  • Mass Spectrometry: statistics & numerical data
  • Models
  • Molecular Sequence Data
  • Molecular Weight
  • Orbitrap
  • Peptide Fragments
  • Peptide Fragments: analysis
  • Peptide Mapping
  • Peptides
  • Peptides: chemistry
  • Peptides: isolation & purification
  • Proteome
  • Proteome: analysis
  • Proteomics
  • Proteomics: methods
  • Proteomics: statistics & numerical data
  • Pseudomonas aeruginosa
  • Pseudomonas aeruginosa: growth & development
  • Pseudomonas aeruginosa: metabolism
  • Reference Standards
  • Saccharomyces cerevisiae Proteins
  • Serum Albumin
  • Software
  • Statistical
  • Tandem Mass Spectrometry
  • acetylation
  • data independent acquisition
  • data-independent acquisition
  • dia
  • epigenetics
  • exposome
  • extramural
  • histone ptm
  • ion trap
  • mass
  • mass spectrometry
  • multiplexing
  • n.i.h.
  • non-u.s. gov't
  • optimization
  • protein adducts
  • proteomics
  • proteomics methods
  • q-exactive
  • quantitative analysis
  • research support
  • saha
  • selected reaction monitoring
  • shotgun
  • spectral correlation
  • spectrometry
  • targeted proteomics
  • toxicoproteomics
  • triple quadrupole

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  • Mikhail Belov

  • Dmitry Grinfeld

  • Philip Compton

  • Neil Kelleher

  • Alexander Makarov

  • Mikhail Belov

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