On-membrane tryptic digestion of proteins for mass spectrometry analysis

Abstract

Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.

Fig. 1

Comparison of MALDI MS spectra obtained from 10 pmol of uroplakin II (UPII) after (a) 30 min on-membrane digestion, (b) 30 min in-gel digestion, (c) 16 h on-membrane digestion, (d) 16 h in-gel digestion, (e) amino acid sequence of bovine mature UPII. The underlined amino acids correspond to the transmembrane domain of the protein. In the spectra, the stars indicate UPII peptides detected after both in-gel and on-membrane digestion, the arrows indicate missed cleavage peptides that appear only after in-gel digestion, and the numbers indicate peptides from the UPII sequence shown in (e) detected only after on-membrane digestion (reproduced from (2) with permission from the American Chemical Society).

Fig. 2

Comparison of MALDI MS spectra obtained from 3 pmol of uroplakin III (UPIII) after (a, c) 30 min on-membrane digestion, (b, d) 30 min in-gel digestion, (e, g) 16 h on-membrane digestion, (f, h) 16 h in-gel digestion, (i) amino acid sequence of bovine UPIII. The underlined amino acids correspond to the transmembrane domain of the protein. In the spectra, the stars indicate UPII peptides detected after both in-gel and on-membrane digestion, the arrows indicate missed cleavage peptides that appear only after in-gel digestion, and the numbers indicate peptides from the UPII sequence shown in (i) detected only after on-membrane digestion. Spectra shown in (a), (b), (e), and (f) were collected in reflectron mode and spectra shown in (c), (d), (g), and (h) in linear mode (reproduced from (2) with permission from the American Chemical Society).