Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry

• Published in: Proteomics (Weinheim) Vol. 2; no. 2; pp. 145 - 150
• Main Authors: Pluskal, Malcolm G., Bogdanova, Alla, Lopez, Mary, Gutierrez, Sara, Pitt, Aldo M.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag GmbH 01.02.2002; WILEY‐VCH Verlag GmbH; Wiley-VCH
• Subjects: Biological and medical sciences; Blood Protein Electrophoresis - methods; Blood Protein Electrophoresis - statistics & numerical data; Blood Proteins - isolation & purification; Electrophoresis, Gel, Two-Dimensional - methods; Electrophoresis, Gel, Two-Dimensional - statistics & numerical data; Fundamental and applied biological sciences. Psychology; Humans; Mass spectrometry; Microbiology; Multiwell; Peptides - isolation & purification; Protein digestion; Proteins - isolation & purification; Proteome - isolation & purification; Sample preparation; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/1615-9861(200202)2:2<145::AID-PROT145>3.0.CO;2-Y

In‐gel peptide digestion has become a widely used technique for characterizing proteins resolved by two‐dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96‐well plate with a low peptide binding membrane and ZipTip® micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.