Workflow for Large Scale Detection and Validation of Peptide Modifications by RPLC-LTQ-Orbitrap: Application to the <i>Arabidopsis thaliana</i> Leaf Proteome and an Online Modified Peptide Library

Post-translational modifications (PTMs) of proteins add to the complexity of proteomes, thereby complicating the task of proteome characterization. An efficient strategy to identify this peptide heterogeneity is important for determination of protein function, as well as for mass spectrometry-based protein quantification. Furthermore, studies of allelic variation or single nucleotide polymorphisms (SNPs) at the proteome level, as well as mRNA editing, are increasingly relevant, but validation and determination of false positive rates are challenging. Here we describe an effective workflow for large scale PTM and amino acid substitution identification based on high resolution and high mass accuracy RPLC-MS data sets. A systematic validation strategy of PTMs using RPLC retention time shifts was implemented, and a decision tree for validation is presented. This workflow was applied to Arabidopsis proteome preparations; 1.5 million MS/MS spectra were processed resulting in 20% sequence assignments, with 5% from modified sequences and matching to 2904 proteins; this high assignment rate is in part due to the high quality spectral data. A searchable modified peptide library for Arabidopsis is available online at http://ppdb.tc.cornell.edu/. We discuss confidence in peptide and PTM assignment based on the acquired data set, as well as implications for quantitative analysis of physiologically induced and preparation-related modifications

Additional file 3: of GSAR: Bioconductor package for Gene Set analysis in R

Source file ‘GSAR_1.9.1.tar.gz’. (GZ 2174 kb

Additional file 2: of GSAR: Bioconductor package for Gene Set analysis in R

Vignette of package GSAR. (PDF 732 kb

Correlation of Relative Abundance Ratios Derived from Peptide Ion Chromatograms and Spectrum Counting for Quantitative Proteomic Analysis Using Stable Isotope Labeling

In this study, S. cerevisiae crude membrane fractions were prepared using the acid-labile detergent RapiGest from cells grown under rich and minimal media conditions using 14N and 15N ammonium sulfate as the sole nitrogen source. Four independent MudPIT analyses of 1:1 mixtures of sample were prepared and analyzed via quantitative multidimensional protein identification technology on a two-dimensional ion trap mass spectrometer. Using the method described in this study, low-abundance integral membrane proteins with up to 14 transmembrane domains were identified and their protein expression determined when sufficient spectrum counting and ion chromatogram information was generated. We demonstrate that spectrum counting and mass spectrometry derived ion chromatograms strongly correlate for determining quantitative changes in protein expression. Spectrum counting proved more reproducible and has a wider dynamic range contributing to the deviation of the two quantitative approaches from a perfect positive correlation

Additional file 1: of GSAR: Bioconductor package for Gene Set analysis in R

Additional document presenting computational considerations and uniqueness of package GSAR. (DOCX 32 kb

PCA plot for colon cancer subtypes.

The number in parentheses indicates the percent of variance, explained by PC component. Only CMS1 is clearly separated from the rest.</p

Protein abundance in ECM receptor interaction pathway for CMS4 and other subtypes.

Protein abundance in ECM receptor interaction pathway for CMS4 and other subtypes.</p

ROAST results.

Pathways differentially expressed between CMS2 and other subtypes.</p

Differences between CMS1 and other CRC subtypes.

A. Protein abundance of common proteins in the intersection of seven KEGG pathway for CMS1. All proteins are part of major histocompatibility complex (MHC) class II. B. Protein abundance in mismatch repair pathway for CMS1 and other subtypes. C. Protein abundance in steroid biosynthesis pathway for CMS1 and other subtypes.</p

ROAST results.

Pathways differentially expressed between CMS4 and other subtypes.</p