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High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel‐separated proteins
Author(s) -
Loo Joseph A.,
Brown Jeffrey,
Critchley Glenn,
Mitchell Charles,
Andrews Philip C.,
Ogorzalek Loo Rachel R.
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:4/5<743::aid-elps743>3.0.co;2-i
Subject(s) - chromatography , chemistry , mass spectrometry , isoelectric focusing , analytical chemistry (journal) , capillary electrophoresis–mass spectrometry , molecular mass , electrospray ionization , matrix assisted laser desorption/ionization , protein mass spectrometry , time of flight mass spectrometry , desorption , ionization , ion , adsorption , biochemistry , organic chemistry , enzyme
The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing‐immobilized pH gradient (IEF‐IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix‐assisted laser desorption/ionization (MALDI)‐MS analysis directly from IEF‐IPG gels. Both ESI and MALDI methods yield sub‐picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI‐MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI‐MS offers high‐throughput analysis of one‐ and potentially two‐dimensional (2‐D) gels. The “virtual 2‐D” gel method with first‐dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2‐D gels. Further sensitivity enhancements for the MALDI‐MS method are provided by post acceleration detection optimized for high mass time‐of‐flight analysis.