Edited by Charles R. Cantor, Sequenom, Inc., San Diego, CA (received for review January 18, 2005)
Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene- and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms/variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics/biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.
↵ * To whom correspondence may be addressed. E-mail: dnedelkov{at}intrinsicbio.com or rnelson{at}intrinsicbio.com.
Author contributions: D.N. designed research; D.N., U.A.K., E.E.N., K.A.T., and R.W.N. performed research; K.A.T. contributed new reagents/analytic tools; D.N., U.A.K., E.E.N., and R.W.N. analyzed data; and D.N. and R.W.N. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: HBS, Hepes-buffered saline; SAA, serum amyloid A.
↵ † It is highly unlikely that the observed protein modifications are a byproduct of the extraction process, because the purification conditions employed in the assay were essentially those traditionally used in protein purification by using immunoprecipitation. It is possible that some of the oxidized protein forms were enhanced by prolonged sample storage at -75°C, but most of the modifications observed here have also been detected in fresh nonfrozen plasma samples (8, 11, 17-20).
↵ ‡ Seven proteins, albumin, cerruloplasmin, C-reactive protein, insulin-like growth factor I, lysozyme, plasminogen, and urine protein 1, were not observed to exhibit any modifications, as indicated by the absence of any other peaks in the mass spectra except for the wild-type protein signal. That is not to say that there were not, in fact, any modifications present in these proteins, especially in albumin, plasminogen, and ceruloplasmin. It should be noted that the method we utilized detailed peptide mapping experiments only in those samples where visible deviations from the wild-type molecular mass were observed in the mass spectra. Because all three of the above-mentioned proteins are high molecular mass proteins, glycosylated (plasminogen and ceruloplasmin), and highly heterogeneous (albumin), the resulting wild-type signals in the mass spectra are relatively wide and of reduced resolution, making the observation of small mass shifts resulting from, e.g., single amino acid substitution, extremely difficult to delineate from the native mass signals.
↵ § The mass spectra resulting from these plasma assays were dominated by signals from the targeted protein. In few instances, signals from nonspecifically bound (to the support) apolipoprotein Cs appeared in the mass spectra. These few additional peaks do not interfere with the overall analysis, as long as their existence is acknowledged and their character (m/z value) is known. Moreover, they can be used advantageously to internally calibrate the mass spectra for a more accurate m/z peak assignment.
Freely available online through the PNAS open access option.
