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Quantitative Analysis of Intact Apolipoproteins in Human HDL by Top-Down Differential Mass Spectrometry

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Quantitative Analysis of Intact Apolipoproteins in Human HDL by Top-Down Differential Mass Spectrometry Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry Matthew T. Mazura,2, Helene L. Cardasisa, Daniel S. Spellmana, Andy Liawb, Nathan A. Yatesa, and Ronald C. Hendricksona,1 aDepartment of Proteomics and bBiometrics Research, Merck Research Labs, 126 E. Lincoln Avenue, P.O. Box 2000, Rahway, NJ 07065 Edited by Fred W. McLafferty, Cornell University, Ithaca, NY, and approved February 17, 2010 (received for review September 22, 2009) Top-down mass spectrometry holds tremendous potential for the shown to provide rich datasets comprised of thousands of peptide characterization and quantification of intact proteins, including in- sequences that can be matched to sequences contained in protein dividual protein isoforms and specific posttranslationally modified databases (3). The routine analysis of mixtures of tryptic peptides forms. This technique does not require antibody reagents and thus by tandem mass spectrometry has resulted in the publication of a offers a rapid path for assay development with increased speci- wide variety of bottom-up applications that include sequence ficity based on the amino acid sequence. Top-down MS is efficient analysis of complete genomes (4, 5) by multidimensional protein whereby intact protein mass measurement, purification by mass identification using strong cation exchange or isoelectric focus- separation, dissociation, and measurement of product ions with ing, compositional analysis of the major components of high- ppm mass accuracy occurs on the seconds to minutes time scale. density lipoprotein (HDL) by spectral counting (6), quantitative Moreover, as the analysis is based on the accurate measurement analysis of metastatic prostate cancer cell lines using stable iso- of an intact protein, top-down mass spectrometry opens a research topes (7), and quantitative analysis of plasma and cerebrospinal paradigm to perform quantitative analysis of “unknown” proteins fluid by differential mass spectrometry (8). It is important to that differ in accurate mass. As a proof of concept, we have applied recognize that because bottom-up methods measure peptides, differential mass spectrometry (dMS) to the top-down analysis of and not proteins, the data may sometimes be degenerate and apolipoproteins isolated from human HDL3. The protein species at cannot distinguish between multiple protein isoforms or distinct 9415.45 Da demonstrates an average fold change of 4.7 (p-value proteins that share a common peptide sequence. 0.017) and was identified as an O-glycosylated form of apolipopro- Kelleher et al. have pioneered “top-down” proteomic techni- tein C-III [NANA-ð2 → 3Þ-Gal-βð1 → 3Þ-GalNAc, þ656.2037 Da], a ques that characterize intact proteins without the use of enzymatic protein associated with coronary artery disease. This work demon- digestion (9, 10). Top-down methods provide a complementary strates the utility of top-down dMS for quantitative analysis of and particularly promising approach for characterizing proteins intact protein mixtures and holds potential for facilitating a better that may exist in multiple forms (11). In a typical top-down experi- understanding of HDL biology and complex biological systems at ment, proteins are introduced into the mass spectrometer directly, the protein level. separated on the basis of their mass-to-charge ratio and then se- quenced from acquired tandem mass spectra. Successful protein quantitative proteomics ∣ Fourier-transfrom mass spectrometry ∣ sequencing can be achieved only when cleavage occurs more or electron transfer dissociation ∣ top-down proteomics ∣ less randomly at the amide backbone and once per molecule so posttranslational modifications that a complete distribution of amide backbone cleavage is ob- served in the corresponding tandem MS spectrum. Classical n nature, mammalian proteins exist as multiple isoforms that threshold dissociation methods [collisionally assisted dissociation Iare derived from variations in the genetic code, alternate splic- (CAD) and infrared multiphoton dissociation] often fail in this ing and processing events, and posttranslational protein modifi- regard (12, 13). Newer low-energy electron-based dissociation cations. The quantification of proteins and protein isoforms in methods electron capture dissociation (ECD) described by Zubar- biological systems is central to expanding the understanding of ev and co-workers (14) and electron-transfer dissociation (ETD) protein function. Antibody-based assays, such as the enzyme- described by Syka and co-workers (15) have opened opportunities linked immune-sorbent assay and the Western blot assay, are for intact protein characterization as the cleavage of the N-Cα the primary tools used to quantify proteins in the laboratory. bond often occurs in a sequence independent manner and pre- In many cases, these tools can provide exquisite sensitivity and serves posttranslational modification (PTMs) (12). Recently, with selectivity due to the availability of well-characterized antibody the introduction of commercial instrumentation that combines reagents that have been carefully developed to selectively bind high-mass resolution and accurate mass measurement capabilities to specific proteins or protein isoforms. A limitation of antibody- with electron-transfer dissociation, it is now possible for industrial based methods is the significant time and effort that is required to generate reagents for each protein studied. When large numbers Author contributions: M.T.M., N.A.Y., and R.C.H. designed research; M.T.M. and H.L.C. of proteins or protein isoforms need to be quantified in a single performed research; M.T.M., N.A.Y., and R.C.H. contributed new reagents/analytic tools; experiment, methods that depend on the availability of selective M.T.M., H.L.C., D.S.S., A.L., N.A.Y., and R.C.H. analyzed data; and M.T.M., H.L.C., D.S.S., antibody reagents become impractical. A.L., N.A.Y., and R.C.H. wrote the paper. Mass spectrometry based proteomics has led to the develop- The authors declare no conflict of interest. ment of established platforms that can detect, identify, and quan- This article is a PNAS Direct Submission. titate thousands of proteins without the use of selective antibody Freely available online through the PNAS open access option. reagents (1, 2). The majority of these techniques utilize proteo- 1To whom correspondence should be addressed. E-mail: [email protected]. lytic enzymes (e.g., trypsin, aspN, and Lys-C) to digest intact 2Present address: Bioanalytical Sciences, Imclone Systems, a Wholly Owned Subsidiary of Eli proteins into short peptides that are ideally suited for analysis Lilly & Co., 22 Imclone Drive, Branchburg, NJ 08876 by mass spectrometry. Collectively, these approaches have been This article contains supporting information online at www.pnas.org/cgi/content/full/ described as “bottom-up” proteomic methods and have been 0910776107/DCSupplemental. 7728–7733 ∣ PNAS ∣ April 27, 2010 ∣ vol. 107 ∣ no. 17 www.pnas.org/cgi/doi/10.1073/pnas.0910776107 Downloaded by guest on September 29, 2021 laboratories to acquire high-resolution full-scan and ETD-MS/MS Many HDL-associated proteins have molecular weights below tandem mass spectra for intact proteins with molecular weights 30 kDa and thus are ideally suited for study by top-down mass in the range of 5–50 kD (16). spectrometry. Here we show that dMS reveals several proteins Differential mass spectrometry (dMS) is a general proteomics quantitatively different in the samples analyzed, one of which workflow that provides relative quantitation from full-scan mass is a protein at 9,415.45 Da that exhibited an increased abundance spectrometry data (17–19). Here, we set out to establish top- in donors having low HDL-c. Protein identification and charac- down dMS capabilities that combine the advantages of intact terization of the intact protein was achieved via ETD liquid chro- protein analysis with quantitation from full spectrum mass spec- matography (LC)-MS/MS data generated on a commercially trometry data (Fig. 1). As a proof-of-concept, we applied dMS to available high-resolution mass spectrometer and analysis by auto- the top-down analysis of HDL isolated from patients having high mated computer-based tools. The intact protein at 9,415.45 Da and low HDL-cholesterol (HDL-c) levels. HDL is one of five was identified by mass spectrometry as an O-glycosylated form major classes of lipoprotein particles in humans. Elevated levels of apolipoprotein C-III [NANA-ð2 → 3Þ-Gal-βð1 → 3Þ-GalNAc, of HDL-c, aka “good cholesterol,” is associated with reduced risk þ656.2037 Da], a protein associated with coronary artery disease of coronary artery disease and hypothesized to be cardioprotec- (25) and demonstrates a unique and potentially promising tive in humans; however, the mechanisms are unknown (20–24). approach for studying the distribution and function of discrete protein isoforms. Collectively these results exhibit the utility of top-down dMS for quantitative analysis of intact protein mixtures Top-down Differential Mass Spectrometry (dMS) and reveal the potential that these antibody-free methods have of Intact Protein Isoforms for expanding the current understanding of HDL biology and complex biological systems at the protein level. High resolution FTMS Legend Results and Discussion Top-Down dMS. Differential mass spectrometry has been success- Low HDL3 High HDL3 fully applied for the relative quantitation of complex peptide mix- tures (17, 18). To adapt dMS to
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