Figure 1

Mass spectrometry workflow. Starting from sample, which could be any preparation of proteins, a work flow could lead to a number of separation/purification techniques or proceed directly to tryptic digestion. Both one- and two-dimensional electrophoresis (1-DE and 2-DE) are used to immunoblot for novel autoantigens followed by analysis, frequently matrix-assisted laser desorption/ionization (MALDI) or surface-enhanced laser desorption/ionization (SELDI), but also including one-dimensional and two-dimensional liquid chromatography (1D-LC and 2D-LC)-tandem mass spectrometry (MS/MS). Complex samples may be separated by affinity chromatography, immunoprecipitation or magnetic bead separation. Techniques may be employed to remove primarily high-abundance proteins, separated based on protein chemistry or interaction kinetics involving immunoglobulins or other proteins. The digested peptides could be subjected to analysis by MALDI, SELDI, and one- or two-dimensional electrospray ionization (ESI)-LC/MS/MS. Gel separated and digested proteins are frequently analyzed with MALDI or SELDI platforms, but are appropriate for ESI as well. More complex sample types, in particular whole cell lysates, which have not been separated, are frequently subjected to one- or two-dimensional LC peptide separation coupled with ESI-MS/MS analysis. The two-dimensional aspect of LC separation allows a greater number of proteins to be identified from very complex samples when compared to the other methods, with the tradeoff being the greatly increased time (10 to 12 hours) to analyze one sample. SELDI-time-of-flight (TOF) and MALDI-TOF are used for profiling while MALDI-TOF-TOF and ESI-MS/MS can sequence the peptides in the sample to positively identify their parent proteins.