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.