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Sample preparation by in-gel digestion for mass spectrometry-based proteomics

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Abstract

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “in-gel digestion” has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches.

Classical in-gel digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel (1). CBB molecules (blue circles) bound to the protein are released by iterative incubation in a buffered organic solvent system (2). To increase digestion efficiency and sequence coverage proteins are reduced (3) and alkylated (4). Proteins are subsequently digested with proteolytic enzymes (scissors symbols), typically trypsin (5). Trypsin cleaves at the amino acid residues arginine (R) and lysine (K). The resulting peptides (A, B, and C) are extracted from the polyacrylamide matrix (6). The peptide solution can be further purified for analysis by mass spectrometry (Section “Concentration and desalting of peptides”)

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Abbreviations

CBB:

Coomassie brilliant blue

CHCA:

α-Cyano-4-hydroxycinnamic acid

CNBr:

Cyanogen bromide

DIGE:

Difference gel electrophoresis

ESI:

Electrospray ionization

FA:

Formic acid

MALDI:

Matrix-assisted laser desorption and ionization

PAGE:

Polyacrylamide gel electrophoresis

PAM-Cys:

Cysteine acrylamide adduct

PMF:

Peptide mass fingerprint

PVDF:

Poly(vinylidene difluoride)

R&A:

Reduction and alkylation

RP:

Reversed phase

TFA:

Trifluoroacetic acid

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  1. Department für Biologie I, Ludwig-Maximilians-Universität, Menzinger Straβe 67, 80638, München, Germany

    Bernhard Granvogl, Matthias Plöscher & Lutz Andreas Eichacker

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  1. Bernhard Granvogl
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  2. Matthias Plöscher
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  3. Lutz Andreas Eichacker
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Correspondence to Lutz Andreas Eichacker.

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Granvogl, B., Plöscher, M. & Eichacker, L.A. Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Anal Bioanal Chem 389, 991–1002 (2007). https://doi.org/10.1007/s00216-007-1451-4

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