Dataset Information

Updated in-gel digestion for protein analysis by mass spectrometry

ABSTRACT: The in-gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub-optimal approaches. We present an updated in-gel digestion protocol. We show that alternative reducing, alkylating agents and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. Our results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and chloroacetamide (CAA) with a subsequent gel wash improve protein identification and sequence coverage, diminish peptide side reactions. Additionally, use of 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid buffer (HEPES) allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updated in-gel digestion protocol described here is highly efficient and offers flexibility to be incorporated in any proteomic laboratory.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Uterine Cervix

DISEASE(S): Cervix Carcinoma

SUBMITTER: Juan Ramon Hernandez-Fernaud

LAB HEAD: Juan Ramon Hernandez Fernaud

PROVIDER: PXD009600 | Pride | 2018-10-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	01_Standardsearch.zip	Other
	02_Acetyl_N-term_peptides.zip	Other
	03_Carbamidomethyl_variable.zip	Other
	Figure_Gel.pptx	Other
	OT_180130_HeLa_DTT_CAA_Met2_Rep_1.raw	Raw

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Publications

Updates of the In-Gel Digestion Method for Protein Analysis by Mass Spectrometry.

Goodman Jennifer K JK Zampronio Cleidiane G CG Jones Alexandra M E AME Hernandez-Fernaud Juan R JR

Proteomics 20181125 23

The in-gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub-optimal approaches. Updates of the in-gel digestion protocol has been presented in the study. It has been shown that alternative reducing, alkylating agent reactions, and tryptic digestion buffers increase peptide and protein identification ...[more]

PMID: 30259661

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