MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

Proteome Science MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration Juraj Lenco 0 1 Renny Lan 0 Nathan Edwards 2 Radoslav Goldman 0 0 Georgetown University, Department of Oncology, Lombardi Comprehensive Cancer Center , 3970 Reservoir Rd NW, Washington, DC 20057 , USA 1 Current address: University Hospital Hradec Kralove; Center for Biomedical Research; Sokolska 581; 500 05 Hradec Kralove; Czech Republic 2 Georgetown University, Department of Biochemistry and Molecular and Cellular Biology , Washington, DC , USA Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not welldeveloped. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma. Native Peptides; denaturing ultrafiltration; MS/MS library; MRM quantitation - Background Proteolysis is an important but perhaps the most overlooked eukaryotic post-translational modification. The biology of neuropeptides [1], peptide hormones [2,3], and unusual proteolytically derived signaling molecules [4,5] stimulates interest in the establishment of appropriate analytical workflows. Mass spectrometry is one of the most useful methods for the analysis of complex peptide mixtures. Proteomic assays typically utilize sequence-specific proteases to characterize the components of complex protein mixtures [6] but the methods for analysis of naturally occurring peptides, without a proteolytic step, are less developed. Applications of mass spectrometry to the study of peptides in various body fluids including cerebrospinal fluid [7], urine [8], synovial fluid [9], saliva [10] and of course serum and plasma [11,12] have been described. A universally useful method for the preparation of the peptides for analysis has not yet emerged and context-specific optimization is typically required. The reported methods include ultrafiltration [13], precipitation by organic solvents [14], solid phase extraction [15], size-exclusion chromatography [11], differential solubilization method [16], and nanoparticle trapping technology [17]. Even methods as simple as direct MALDI-TOF analysis of a complex mixture in a body fluids were used successfully [12] with the benefit of high-throughput, minimal preparative losses of analytes, and minimal sample requirements. Indeed, MALDI- or SELDI-TOF based analyses are most likely the richest source of information about native peptides. On the other hand, these methods suffer from inherent quantitative limitations [15,18]. The original semi quantitative screens are therefore followed by the development of isotope dilution kinetic assays [19] and, most recently, multiple reaction monitoring (MRM) LC-MS/MS quantification of target peptides [20]. MRM has emerged as an LC-MS alternative to antibody based assays for accurate protein quantification [21]. This targeted technology for monitoring of select proteins in complex matrices exploits the sensitivity and selectivity of triple quadrupole mass spectrometers. Specific combinations of precursor m/z and its fragments (called transitions) are monitored with linear quantification across several orders of magnitude. Perhaps the most valuable feature of the assays is the ability to multiplex hundreds of analytes [22]. The design and validation of new MRM assays, however, is labor and cost intensive if information about the fragmentation of the peptides of interest is not available. For protein quantification using a tryptic peptide, empirical data stored in PeptideAtlas may be exploited [23]. PeptideAtlas lists peptides most frequently observed in proteomic studies and their most abundant fragments. Unfortunately, such an information-rich database is not available for native peptides and minimal data applicable to ESI ionization are publically available. This substantially increases the number of potential transitions that must be considered in assay design, to account for multiple precursor charge states, the high number of potential fragments, and their different charge states. Our overall goal was to develop a workflow leading from standardized preparation of serum samples, through data management facilitating the design of MRM quantification, to the validation in a clinically applicable assay. We have therefore developed and characterized a simple method that efficiently inhibits proteolytic processes in body fluids, with sufficient peptide recovery for LC-MRM MS quantification. To facilitate the design of the MRM assays, we established a Skyline [24] library of MS/MS spectra of native serum peptides. To our best knowledge, this is the first attempt to use 2D-HPLC ESI-MS/MS to build a MS/MS library of native peptides in order to facilitate the design of quantitative MRM assays. As a proof-of-concept, the library was applied to the MRM assay development for three peptides with diagnostic potential in liver cirrhosis and hepatocellular cancer [25-27]. This workflow is not specific to hepatocellular cancer, and is expected to facilitate the development of quantitative LC-MS-MRM assays of native peptides in other studies as well. Specific experimental endpoints may require different preparative steps but we believe that the establishment of a publically available library of MS/MS spectra will facilitate rapid screening of target analytes in clinically relevant samples. This is expected to improve the ability of researchers to validate previously suggested or newly discovered biomarker candidates. Results and Discussion Quantification of peptides in biological samples is of general interest [2,5,11]. The mass spectrometric MRM quantification of peptides has become a viable alternative to the traditional ELISA assays [28,29]. MRM quantification is an appealing alternative particularly for multiplex assays of proteolytically modified peptides, such as the native peptides discussed in this article. A discussion of the biology of the native peptides is beyond the scope of this paper; we do not make any claims as to physiological or diagnostic relevance of the peptides analyzed here. We focus on the presentation of a general method for peptide extraction from body fluids and on the generation of native peptide MS/MS library to facilitate the design of quantitative assays for the exploration of the biology of native peptides. It is hard to imagine that a specific quantitative ELISA assay for each of the peptides can (...truncated)