Optimization of separation and digestion conditions in immune complexome analysis

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.