Recovery of gel-separated proteins for in-solution digestion and mass spectrometry

A. P. Jonsson, Y. Aissouni, C. Palmberg, Piergiorgio Percipalle, E. Nordling, B. Daneholt, H. Jörnvall, T. Bergman

Research output: Contribution to journalArticle

Abstract

A protocol for mass spectrometry of gel-separated proteins resulting in significantly increased sequence coverage and in improved possibilities for detection and identification of posttranslational modifications was developed. In relation to the standard in-gel digestion procedure, the sequence coverage using a combination of matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry was on the average increased by 30%. The method involves electroblotting of the gel-separated proteins to a poly(vinylidene difluoride) membrane. The proteins are extracted from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile and lyophilized. After reconstitution of the protein extract in digestion buffer, proteolytic cleavage is carried out in-solution as opposed to the standard in-gel digestion procedure. This allows recovery of large and hydrophobic peptides for mass spectrometry and reduces the risk for entrapment of proteolytic peptides in the gel matrix. The method was applied to proteins in the 30-40-kDa range with highly different structural properties. The improved ability to localize and determine protein modifications is shown for N-terminal acetylation and methylation of a histidine residue. Furthermore, the method enables fast screening of homologous protein sequences.

Original languageEnglish (US)
Pages (from-to)5370-5377
Number of pages8
JournalAnalytical Chemistry
Volume73
Issue number22
DOIs
StatePublished - Nov 15 2001

Fingerprint

Mass spectrometry
Gels
Recovery
Proteins
Membranes
Trifluoroacetic Acid
Acetylation
Electrospray ionization
Peptides
Methylation
Histidine
Ionization
Structural properties
Desorption
Buffers
Screening
Lasers

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Jonsson, A. P., Aissouni, Y., Palmberg, C., Percipalle, P., Nordling, E., Daneholt, B., ... Bergman, T. (2001). Recovery of gel-separated proteins for in-solution digestion and mass spectrometry. Analytical Chemistry, 73(22), 5370-5377. https://doi.org/10.1021/ac010486h

Recovery of gel-separated proteins for in-solution digestion and mass spectrometry. / Jonsson, A. P.; Aissouni, Y.; Palmberg, C.; Percipalle, Piergiorgio; Nordling, E.; Daneholt, B.; Jörnvall, H.; Bergman, T.

In: Analytical Chemistry, Vol. 73, No. 22, 15.11.2001, p. 5370-5377.

Research output: Contribution to journalArticle

Jonsson, AP, Aissouni, Y, Palmberg, C, Percipalle, P, Nordling, E, Daneholt, B, Jörnvall, H & Bergman, T 2001, 'Recovery of gel-separated proteins for in-solution digestion and mass spectrometry', Analytical Chemistry, vol. 73, no. 22, pp. 5370-5377. https://doi.org/10.1021/ac010486h
Jonsson AP, Aissouni Y, Palmberg C, Percipalle P, Nordling E, Daneholt B et al. Recovery of gel-separated proteins for in-solution digestion and mass spectrometry. Analytical Chemistry. 2001 Nov 15;73(22):5370-5377. https://doi.org/10.1021/ac010486h
Jonsson, A. P. ; Aissouni, Y. ; Palmberg, C. ; Percipalle, Piergiorgio ; Nordling, E. ; Daneholt, B. ; Jörnvall, H. ; Bergman, T. / Recovery of gel-separated proteins for in-solution digestion and mass spectrometry. In: Analytical Chemistry. 2001 ; Vol. 73, No. 22. pp. 5370-5377.
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