Because of dynamic range, reproducibility and throughput limitations, 2D gel electrophoresis was surpassed by bottom-up and top-down mass spectrometry approaches for decoding protein modifications. In bottom-up approaches, proteins are digested with trypsin, and selected reaction monitoringor multiple reaction monitoringmass spectrometry is used to detect peptidesthat contain the protein modifications. Osteopontin splice variants were identified and quantified using MRM-MS ; novel proteoforms of prostate specific antigen were also identified in clinical samples via MRM-MS. In a larger population study, the concentration of three selected single amino acid polymorphism peptides, representing the Complement Component C7, Complement Factor H, and Complement Component C5 proteins, were measured by SRM-MS from 290 individuals. In another population study, serum peptide variations were studied in 500 healthy individuals using a regular MS analysis, but the peptides detected were exopeptidase products derived from relatively abundant serum proteins. Top-down MS approaches provide more accurate and complete results for protein variants identification because 
there is no Cefetamet pivoxil HCl prerequisite for a priori knowledge of the protein modification in order to select the appropriate modification-specific peptide. But analyzing and quantifying intact proteins and their modifications with mass spectrometry can be challenging. Our group has devised a simple method termed mass spectrometric immunoassaythat combines a one-step affinity protein isolation with MS analysis that is ideally suited for high-throughput analysis of human plasma proteins and their variants. Antibodies are surface-immobilized in small, porous microcolumns that are fitted at the entrance of a pipettor tip. Samples are passed through the pipette tip repetitively until enough protein is bound to the antibody. Following washing with buffer and/or other mild solutions to remove non-specifically bound sample components, the proteins are eluted with a small volume of matrix solution and deposited directly onto a MALDI target for ensuing MS analysis. We’ve applied this approach to investigate the diversity of 25 human plasma proteins from a cohort of 96 healthy individuals, resulting in the detection of 76 structural variants, each occurring with different frequencies. Subsequently, we’ve screened 1,000 individuals from four geographical regions in the United States, and determined the variants and their frequencies for five proteins – beta-2-microglobulin, cystatin C, retinol binding protein, transferrin, and transthyretin. The qualitative data from those studies provided a first glimpse into the extent of protein structural diversity in the general population. Recently we have Albaspidin-AA developed and validated fully quantitative mass spectrometric immunoassays for 4 of those clinically relevant proteins – beta-2-microglobulin, cystatin C, retinol binding proteinand transthyretin. Beta-2-microglobulin is used in the diagnosis of active rheumatoid arthritis and kidney disease, and a structural variant of b2m has been associated with autoimmune disease and small-cell lung cancer. Cystatin C is a serine proteinase inhibitor with implications in renal failure. Retinol binding protein has also been implicated in renal disease, with the increased presence of its truncated variants suggested as indication of renal failure.