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Enhancing proteome coverage by using strong anion exchange in tandem with basic-pH reversed-phase chromatography for sample multiplexing-based proteomics

ABSTRACT: Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three datasets, one using only BPRP fractionation, and two others of each SAX-partition followed by BPRP. The three datasets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition dataset contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines investigated herein.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD044393 | Pride | 2023-12-28

REPOSITORIES: Pride

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Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for Sample Multiplexing-Based Proteomics.

Zhang Tian T Liu Xinyue X Rossio Valentina V Dawson Shane L SL Gygi Steven P SP Paulo Joao A JA

Journal of proteome research 20231114 8

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior ...[more]

PMID: 37962907

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			Action	DRS
	2023-10-13_RAW_files.xlsx	Xlsx
	PeptideTablesOnPRIDE.xlsx	Xlsx
	ea12864.raw	Raw
	ea12864_ManyCell_ABC.mzIdentML	Mzid
	ea12865.raw	Raw