Journal ArticleDOI
Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.
TLDR
The combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS and 1,504 yeast proteins were unambiguously identified in this single analysis.Abstract:
Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC−MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 μm i.d.) approach for RP-LC−MS/MS analysis. More than 162 000 MS/MS spectra were collected with 26 815 matched to yeast peptides (7537 unique peptides). A total of 1504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. ...read more
Citations
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Journal ArticleDOI
Mass spectrometry-based proteomics
Ruedi Aebersold,Matthias Mann +1 more
TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Journal ArticleDOI
Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry
Joshua E. Elias,Steven P. Gygi +1 more
TL;DR: This work clarifies the preferred methodology by addressing four issues based on observed decoy hit frequencies: the major assumptions made with this database search strategy are reasonable, concatenated target-decoy database searches are preferable to separate target and decoydatabase searches, and the theoretical error associated with target-Decoy false positive (FP) rate measurements can be estimated.
Journal ArticleDOI
Semi-supervised learning for peptide identification from shotgun proteomics datasets
TL;DR: An algorithm, called Percolator, is described, for improving the rate of confident peptide identifications from a collection of tandem mass spectra, using semi-supervised machine learning to discriminate between correct and decoy spectrum identifications.
Journal ArticleDOI
Comparing protein abundance and mRNA expression levels on a genomic scale
Dov Greenbaum,Christopher M. Colangelo,Christopher M. Colangelo,Kenneth R. Williams,Kenneth R. Williams,Mark Gerstein +5 more
TL;DR: This work merges many of the available yeast protein-abundance datasets, using the resulting larger 'meta-dataset' to find correlations between protein and mRNA expression, both globally and within smaller categories.
Journal ArticleDOI
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS
TL;DR: The AQUA strategy was used to quantify low abundance yeast proteins involved in gene silencing, quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay.
References
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Journal ArticleDOI
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Journal ArticleDOI
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Proteomics to study genes and genomes
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Journal ArticleDOI
The current state of two-dimensional electrophoresis with immobilized pH gradients.
Angelika Görg,Christian Obermaier,Günther Boguth,Alois Harder,Burghardt Scheibe,Robert Wildgruber,Walter Weiss +6 more
TL;DR: The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) is updated in this article, and the results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given.