title: Optimization of separation and digestion conditions in immune complexome analysis.
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.
authors.affiliation: Course of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan.
authors.forename: Miyako
authors.initials: M
authors.lastname: Baba
authors.forename: Kaname
authors.initials: K
authors.lastname: Ohyama
authors.forename: Naoya
authors.initials: N
authors.lastname: Kishikawa
authors.forename: Naotaka
authors.initials: N
authors.lastname: Kuroda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006736:Horses; D006801:Humans; D008872:Microwaves; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000936:Antigen-Antibody Complex; D014357:Trypsin
keywords: Gradient elution time; Immune complexome analysis; Microwave-assisted digestion; Nano-liquid chromatography–tandem mass spectrometry; Tryptic digestion