Cedarville University DigitalCommons@Cedarville Pharmaceutical Sciences Faculty Publications Department of Pharmaceutical Sciences 6-2004 Analysis of the Human Serum Proteome King C. Chan David A. Lucas Denise Hise Carl F. Schaefer Zhen Xiao See next page for additional authors Follow this and additional works at: https://digitalcommons.cedarville.edu/ pharmaceutical_sciences_publications Part of the Pharmacy and Pharmaceutical Sciences Commons This Article is brought to you for free and open access by DigitalCommons@Cedarville, a service of the Centennial Library. It has been accepted for inclusion in Pharmaceutical Sciences Faculty Publications by an authorized administrator of DigitalCommons@Cedarville. For more information, please contact [email protected]. Authors King C. Chan, David A. Lucas, Denise Hise, Carl F. Schaefer, Zhen Xiao, George M. Janini, Kenneth H. Buetow, Haleem J. Issaq, Timothy D. Veenstra, and Thomas P. Conrads Clinical Proteomics Journal Copyright ©Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/04/01:101–225/$25.00 Serum/Plasma Proteome Analysis of the Human Serum Proteome King C. Chan,1,† David A. Lucas,1,† Denise Hise,2 Carl F. Schaefer,2 Zhen Xiao,1 George M. Janini,1 Kenneth H. Buetow,2 Haleem J. Issaq,1 Timothy D.Veenstra,1 and Thomas P. Conrads1,* 1Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick, SAIC-Frederick, Inc, PO Box B, Frederick, MD 21702 2Center for Bioinformatics, National Cancer Institute, Bethesda, MD 20892 †These authors contributed equally to this work. each of which was analyzed by microcapillary Abstract reversed-phase liquid chromatography coupled Changes in serum proteins that signal online with MS/MS analysis. histopathological states, such as cancer, are useful This investigation resulted in the identifica- diagnostic and prognostic biomarkers. Unfortu- tion of 1444 unique proteins in serum. Proteins nately, the large dynamic concentration range from all functional classes, cellular localization, of proteins in serum makes it a challenging and abundance levels were identified. proteome to effectively characterize. Typically, This study illustrates that a majority of lower methods to deplete highly abundant proteins abundance proteins identified in serum are pre- to decrease this dynamic protein concentra- sent as secreted or shed species by cells as a result tion range are employed, yet such depletion of signalling, necrosis, apoptosis, and hemolysis. results in removal of important low abundant These findings show that the protein content of proteins. serum is quite reflective of the overall profile of Amulti-dimensional peptide separation strat- the human organism and a conventional multi- egy utilizing conventional separation techniques dimensional fractionation strategy combined with combined with tandem mass spectrometry MS/MS is entirely capable of characterizing a sig- (MS/MS) was employed for a proteome analysis nificant fraction of the serum proteome. We have of human serum. Serum proteins were digested constructed a publicly available human serum with trypsin and resolved into 20 fractions by proteomic database (http://bpp.nci.nih.gov) to ampholyte-free liquid phase isoelectric focusing. provide a reference resource to facilitate future These 20 peptide fractions were further fraction- investigations of the vast archive of pathophysio- ated by strong cation-exchange chromatography, logical content in serum. *Author to whom all correspondence and reprint requests should be addressed: E-mail: [email protected] 101 102 ________________________________________________________________________________ Chan et al. Key Words: Serum; proteomics; mass spectrome- to correlate data obtained from tandem MS try; multi-dimensional separation; isoelectric (MS/MS) measurements for protein identifi- focusing; biomarker. cation. Because a potential archive of patho- Introduction physiological information is endowed to serum from its constant perfusion of tissues, Amajor trend underlying current biological particular focus has been given to developing research is the development and application methods to allow its facile investigation by of analytical methods capable of making proteomic technologies. Serum is a rather global measurements of entire biological sys- unique biological sample in that no specific tems. These advances have created unique cellular genomic expression per se contributes opportunities in the field of medicine, where to its protein content, but rather it may be the results from gene expression studies are hypothesized that the protein content is con- expected to help identify cellular alterations tributed by the summation of all cellular associated with disease diagnosis, etiology, genomic expression in the organism. It is progression, outcome, and response to ther- therefore clear that the collection of histo- apy. A major goal is to obtain a greater under- pathological information in serum is com- standing of the function of proteins in a prised not only of the expected circulatory cellular context, as well as their more con- proteins such as immunoglobulins, but also of ventionally delineated individual molecular peptides and proteins that are secreted into function. This global view promises to provide the blood and shed species from diseased, a greater understanding of the cellular responses dying, or dead cells (2). to events such as cell division, differentiation, The importance of characterizing the protein respiration, hormonal signalling, and changes content of serum is underscored by the fact in homeostasis. The impetus for conducting that subtle changes of low abundance proteins global measurements of biological systems is have been shown to indicate development of to establish new diagnostic approaches and different disease states such as ovarian (3,4), therapeutic targets for a host of maladies, breast, (5) and prostate (6,7) cancer. Despite including infectious diseases, behavioral dis- decades of research, however, only a few hun- orders, developmental defects, neurodegener- dred proteins have been identified from either ative diseases, aging, and cancer. serum or plasma (8,9). Unfortunately, the large Whereas the availability of complete genome dynamic concentration range of proteins in sequences opens the door to important biologi- serum renders this proteome cumbersome to cal advances, much of the detailed under- effectively characterize. Most serum proteome standing of cellular systems and the roles of studies have therefore removed high abun- its constituents will, by necessity, be based dance proteins, such as albumin or immuno- upon proteomics, which has classically been globulins, prior to analysis. It is plausible that defined as the study of the entire complement depletion of highly abundant proteins from of proteins, and their modifications, expressed serum may in fact result in concomitant deple- by a cell. Typically, proteomic investigations tion of the histopathological archive of poten- are conducted using mass spectrometry (MS) tially important peptides and proteins that as the core analytical technique for protein may be present at low abundance. Hence, the identification and relative quantitation (1). multi-dimensional chromatographic-MS/MS Current MS-based proteomic measurements method we have utilized in the present work rely heavily on databases of predicted proteins does not rely on the depletion of any highly generated from genome sequence information abundant proteins. Clinical Proteomics _______________________________________________________________ Volume 1, 2004 Analysis of the Human Serum Proteome _______________________________________________________ 103 While the importance of studying the serum were identified, demonstrating the ability to proteome is clear, its composition and com- conduct investigations of human serum using plexity make it one of the most challenging conventional proteomic technologies. proteome samples to characterize. Serum is composed of a wide variety of biomolecules Materials ranging from large molecules such as pro- 1. Pooled standard human serum, NH4HCO3, teins and lipids, to small metabolites such as NaHCO2, urea, formic acid, trifluoroacetic peptides, amino acids, and electrolytes. Twenty- acid (TFA), and dithiothreitol (DTT) were two proteins, such as albumin, immunoglobu- purchased from Sigma (St. Louis, MO). lins, haptoglobin, transferrin, and lipoproteins, 2. Porcine sequencing grade modified trypsin account for 99% of the protein mass of serum was purchased from Promega (Madison, WI). (2). It is estimated that as many as 10,000 unique 3. High performance liquid chromatography proteins are present within the human serum (HPLC) grade acetonitrile was obtained proteome that span a dynamic range of concen- from EMD Chemicals Inc. (Gibbstown, NJ). tration estimated to be greater than 109 (2). This 4. All buffers and reagents were used as sup- dynamic range of protein concentration is the plied from the manufacturer and prepared primary factor that makes global characteriza- in double distilled water using a NANOP- tion of the serum proteome so challenging and ure Diamond water system (Barnstead International, Dubuque, IA). necessitates effective fractionation methods capable of separating high abundance proteins Methods from low abundance species prior to MS or MS/MS analyses. Serum Tryptic Digestion The most extensive characterization and Two hundred microliters of a pooled stan- cataloging