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Optimization of separation and digestion conditions in immune complexome analysis


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Title: Optimization of separation and digestion conditions in immune complexome analysis
Authors: Baba, Miyako / Ohyama, Kaname / Kishikawa, Naoya / Kuroda, Naotaka
Issue Date: 2013
Publisher: Elsevier Inc.
Citation: Analytical Biochemistry, 443(2), pp.181-186; 2013
Abstract: Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS). To improve this method, we initially investigated the effects of two factors—the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)—on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25–400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time.
Keywords: Gradient elution time / Immune complexome analysis / Microwave-assisted digestion / Nano-liquid chromatography–tandem mass spectrometry / Tryptic digestion
URI: http://hdl.handle.net/10069/33939
ISSN: 00032697
DOI: 10.1016/j.ab.2013.08.026
Rights: © 2013 Elsevier Inc. All rights reserved.
Type: Journal Article
Text Version: author
Appears in Collections:Articles in academic journal

Citable URI : http://hdl.handle.net/10069/33939

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