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.

One of the major difficulties in mining low abundance biomarkers from serum or plasma is due to the fact that a small number of proteins such as albumin, alpha2-macroglobulin, transferrin, and immunoglobulins, may represent as much as 80% of the total serum protein. The large quantity of these proteins makes it difficult to identify low abundance proteins in serum using traditional 2-dimensional electrophoresis. We recently used a combination of multidimensional liquid chromatography and gel electrophoresis coupled to matrix-assisted laser desorption/ionization-quadrupole-time of flight and Ion Trap liquid chromatography-tandem mass spectrometry to identify protein markers in sera of Alzheimer's disease (AD), insulin resistance/type-2 diabetes (IR/D2), and congestive heart failure (CHF) patients. We identified 8 proteins that exhibit higher levels in control sera and 36 proteins that exhibit higher levels in disease sera. For example, haptoglobin and hemoglobin are elevated in sera of AD, IR/D2, and CHF patients. The levels of several other proteins including fibrinogen and its fragments, alpha 2-macroglobulin, transthyretin, pro-platelet basic protein, protease inhibitors clade A and C, as well as proteins involved in the classical complement pathway such as complement C3, C4, and C1 inhibitor, were found to differ between IR/D2 and control sera. The sera levels of proteins, such as the 10 kDa subunit of vitronectin, alpha 1-acid glycoprotein, apolipoprotein B100, fragment of factor H, and histidine-rich glycoprotein were observed to be different between AD and controls. The differences observed in these biomarker candidates were confirmed by Western blot and the enzyme-linked immunosorbent assay. The biological meaning of the proteomic changes in the disease states and the potential use of these changes as diagnostic tools or for therapeutic intervention will be discussed.