Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/ml range with percent coefficients of variation from 3 to 15 % without immunoaffinity enrichment of either proteins

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage,

BACKGROUND: Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass. CONTENT: We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 mol/L on a molar basis or from about 50 000 to 1 mg/L mass abundance.

Experience from different fields of life sciences suggests that accessible, complete reference maps of the components of the system under study are highly beneficial research tools. Examples of such maps include libraries of the spectroscopic properties of molecules, or databases of drug structures in analytical or forensic chemistry. Such maps, and methods to navigate them, constitute reliable assays to probe any sample for the presence and amount of molecules contained in the map. So far, attempts to generate such maps for any proteome have failed to reach complete proteome coverage 1-3 . Here we use a strategy based on high-throughput peptide synthesis and mass spectrometry to generate an almost complete reference map (97% of the genome-predicted proteins) of the Saccharomyces cerevisiae proteome. We generated two versions of this mass-spectrometric map, one supporting discovery-driven (shotgun) 3,4 and the other supporting hypothesis-driven (targeted) 7 , which requires precise measurement of the same set of peptides over a large number of samples. Protein measurements over 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, influencing the levels of related proteins. Our results suggest that selective pressure favours the acquisition of sets of polymorphisms that adapt protein levels but also maintain the stoichiometry of functionally related pathway members. In proteomics, the generation of reference maps covering a complete proteome has been attempted in two ways. The first is based on the development of immunoassays to detect target proteins and is exemplified for the human proteome by the Protein Atlas project 8 . The second approach is in-depth mapping of a proteome through the collection of fragment ion spectra from multiple mass-spectrometrybased shotgun proteomic experiments We next used these peptides to generate two reference spectral libraries, each one supporting a commonly used proteomic method. We analysed the peptide set on a linear ion trap (LIT)-type instrument (LIT-Orbitrap hybrid) to generate reference fragment ion spectra for spectral matching of data acquired in discovery experiments. We used a QQQ-type mass spectrometer (QTRAP hybrid) operated in the SRM-triggered tandem mass spectrometry (MS/MS) mode with fragmentation in the second quadrupole 17 to generate fragment ion spectra for the extraction of optimal SRM coordinates for targeted measurement of specific proteins To maximize proteome coverage, we combined our LIT data with quality-filtered LIT data from yeast extracts that had been submitted to PeptideAtlas 1 and consensus spectra from the National Institute of Standards and Technology (NIST) yeast ion-trap spectral library

A “bottom-up ” proteomics approach and a two-dimen-sional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracel-lular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identi-fied in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 g/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellu-lar localization was determined based on Genome Ontol-ogy terms for all the 1,139 proteins of which 34 % were

Analysis of primary animal and human tissues is key in biological and biomedical research. Comparative pro-teomics analysis of primary biological material would benefit from uncomplicated experimental work flows ca-pable of evaluating an unlimited number of samples. In this report we describe the application of label-free pro-teomics to the quantitative analysis of five mouse core proteomes. We developed a computer program and nor-malization procedures that allow exploitation of the quan-titative data inherent in LC-MS/MS experiments for rela-tive and absolute quantification of proteins in complex mixtures. Important features of this approach include (i) its ability to compare an unlimited number of samples, (ii) its applicability to primary tissues and cultured cells, (iii) its straightforward work flow without chemical reaction

A SISCAPA (stable isotope standards and capture by antipeptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM 1 detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ϳ1eϩ4, and the ability to multiplex measurements of hundreds of peptides per sample (1). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications. An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ϳ1 g of total peptide sample, i.e. the product obtained from digesting ϳ14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1-20 g/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1). The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ϳ85% of the protein mass (2) and results in an increase of ϳ7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source. An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4). In its initial implementation, SISCAPA used very small (ϳ10-nl) columns of POROS chro-

The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometrybased assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the ϳ20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.

forms have resulted in the generation of large num-bers of candidate cancer biomarkers, a comparable system for subsequent quantitative assessment and verification of all candidates is lacking. Established immunoassays and available antibodies permit anal-ysis of small subsets of candidates; however, the lack of commercially available reagents, coupled with high costs and lengthy production and purification times, have rendered the large majority of candidates untestable. CONTENT: Mass spectrometry (MS), and in particular multiple reaction monitoring (MRM)-MS, has emerged as an alternative technology to immunoas-says for quantification of target proteins. Novel bio-markers are expected to be present in serum in the

The growing use of selected reaction monitoring (SRM) mass spectrometry in proteomic analyses led us to inves-tigate how to identify peptides by SRM using only a min-imal number of fragment ions. By using a computational model of the SRM work flow we computed the potential interferences from other peptides in a given proteome. From these results, we selected the deterministic SRM addresses that contained sufficient information to confer peptide and protein identity that we termed unique ion signatures (UIS). We computationally showed that UIS comprised of only two transitions are diagnostic for>99 % of Escherichia coli proteins and>96 % of human proteins that possess a sequence-unique peptide. We demonstrated an example of experimental use of UIS us-ing a modified SRM methodology to profile the E. coli tricarboxylic acid cycle from a single injection of cell ly-