The MALDI-TOF spectra of peptides from the sera of normal and myocardial infarction patients produced patterns that provided an accurate diagnostic of MI. In myocardial infarction, the spectral pattern originated from the cleavage of complement C3 alpha chain to release the C3f peptide and cleavage of fibrinogen to release peptide A. The fibrinogen peptide A and complement C3f peptide were in turn progressively truncated by aminopeptidases to produce two families of fragments that formed the characteristic spectral pattern of MI. Time course and inhibitor studies demonstrated that the peptide patterns in the serum reflect the balance of disease-specific-protease and aminopeptidase activity ex vivo.

Electrophoretic and chromatographic sample preparations were compared and together detected the presence of some 600 types of protein products in human serum. Proteins from crude serum preseparated by ionic electrophoresis, chromatography, or a combination of both were analyzed. Proteins were digested with trypsin or chymotrypsin. Naturally occurring peptides were also collected by reversed-phase chromatography. The resulting peptides were identified by tandem mass spectrometry. The peptides were either desorbed by a laser from a metal chip into a quadrupole-time-of-flight mass spectrometer or ionized as an electro-spray from reversed-phase chromatography via a metal needle under voltage into an ion-trap mass spectrometer. All of the commonly known proteins associated with serum were detected, and the two mass spectrometers agreed on the identity of abundant serum proteins. Preseparation of serum proteins prior to digestion markedly enhanced the capacity to detect un-common proteins from blood. Electrophoretic- and chromatography-based experiments were found to be complementary. Many novel cellular proteins not previously associated with serum were recorded.

Many proteomics studies are limited to the identification of only the most abundant proteins in a sample due to the high sample complexity in most proteomes. We have here addressed this problem by prefractionation of human blood samples using microchromatography. We show that our approach resulted in high-stringency tryptic peptides identified by LC-ESI-MS/MS. Serum proteins were fractionated by batch and stepwise preparative chromatography using various types of chromatography resins (propyl sulfate, quaternary amine, diethylaminoethanol, cibachron blue, phenol Sepharose, carboxy methyl sepharose, hydroxyl apatite, heparin, concanavalin A and protein G) that were compared. The efficacy of sample fractionation was determined by protein assays, electrophoresis, and mass spectrometry. Tryptic peptides were separated by C18 liquid chromatography with electrospray ionization via metal needle at 2 microL/min with ion trap tandem mass spectrometry. The MS/MS spectra were correlated to some 4396 distinct sequences of the human forward RefSeq by X!TANDEM. Of these, 61% have been detected by other algorithms, but 3219 (73%) were never previously reported from blood by X!TANDEM. The use of a simple apparatus for making gravity microchromatography columns that permits the rapid side-by-side fractionation of many serum samples is described. Disposable microcolumns rapidly prepared blood samples for LC ESI-MS/MS that detected both tissue and cell leakage proteins known to exist in the approximately 1 ng/mL range and some circulating receptor sequences. Our results demonstrate that the depletion of albumin or IgG was not necessary prior to LC-MS/MS and that multiple forms of protein chromatography will be useful for complete identification of blood proteins.