Proteome Analysis Using Gel-LC-MS/MS

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data a...

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Vydané v:Current protocols in protein science Ročník 96; číslo 1; s. e93
Hlavní autori: Goldman, Aaron R, Beer, Lynn A, Tang, Hsin-Yao, Hembach, Peter, Zayas-Bazan, Delaine, Speicher, David W
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States 01.06.2019
Predmet:
Chemical Fractionation
Chromatography, High Pressure Liquid - methods
Electrophoresis, Polyacrylamide Gel
High-Throughput Screening Assays - instrumentation
High-Throughput Screening Assays - methods
Peptides - chemistry
Proteome - analysis
Tandem Mass Spectrometry
proteome fractionation
LC-MS/MS
proteomics
in-gel digestion
gel-LC-MS/MS
mass spectrometry
SDS gels
ISSN:1934-3663, 1934-3663
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Shrnutí:This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.
Bibliografia:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:1934-3663
1934-3663
DOI:10.1002/cpps.93