Online microwave D-cleavage LC-ESI-MS/MS of intact proteins: site-specific cleavages at aspartic acid residues and disulfide bonds

Journal of Proteome Research
Nicolas Hauser, Franco Basile

Abstract

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for d...Continue Reading

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Citations

Jul 14, 2010·Amino Acids·Bjoern MeyerMichael Karas
Jan 2, 2010·Journal of the American Society for Mass Spectrometry·P Muralidhar ReddyYen-Peng Ho
Apr 19, 2011·International Journal of Mass Spectrometry·Catherine FenselauStephen Swatkoski
Apr 3, 2015·Journal of the American Society for Mass Spectrometry·Lee Chuin ChenKenzo Hiraoka
Apr 1, 2020·Antibodies·Georg TscheuschnerMichael G Weller
Mar 28, 2008·Journal of Proteome Research·Nicolas J HauserFranco Basile
Oct 14, 2010·Analytical Chemistry·Ning LiuZongwei Cai

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