title: Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry.
abstract: Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL(3). The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 --> 3)-Gal-beta(1 --> 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.
authors.affiliation: Department of Proteomics, Merck Research Labs, 126 E Lincoln Avenue, PO Box 2000, Rahway, NJ 07065, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Mazur
authors.forename: Helene L
authors.initials: HL
authors.lastname: Cardasis
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Spellman
authors.forename: Andy
authors.initials: A
authors.lastname: Liaw
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Yates
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Hendrickson
mesh_terms: D000595:Amino Acid Sequence; D053305:Apolipoprotein C-III / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D008076:Cholesterol, HDL / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D053305:Apolipoprotein C-III; D008076:Cholesterol, HDL; D020033:Protein Isoforms
references.citation: Proteomics. 2006 Apr;6(7):2101-7
references.pmid: 16518871
references.citation: Science. 1999 May 21;284(5418):1289-90
references.pmid: 10383309
references.citation: Annu Rev Biochem. 2001;70:437-73
references.pmid: 11395414
references.citation: Anal Chem. 2004 Oct 15;76(20):6085-96
references.pmid: 15481957
references.citation: Nat Methods. 2007 Oct;4(10):817-21
references.pmid: 17901871
references.citation: Ann Med. 2009;41(1):11-8
references.pmid: 19085339
references.citation: Anal Biochem. 1985 Oct;150(1):76-85
references.pmid: 3843705
references.citation: Nature. 2007 Dec 13;450(7172):991-1000
references.pmid: 18075578
references.citation: J Proteome Res. 2010 Mar 5;9(3):1392-401
references.pmid: 20095649
references.citation: Int J Mass Spectrom. 2007 Jan 1;259(1-3):197-203
references.pmid: 17364019
references.citation: Proc Natl Acad Sci U S A. 1965 Dec;54(6):1699-705
references.pmid: 5218921
references.citation: J Clin Invest. 2007 Mar;117(3):746-56
references.pmid: 17332893
references.citation: J Lipid Res. 1981 Feb;22(2):339-58
references.pmid: 6787159
references.citation: Science. 2008 Dec 12;322(5908):1702-5
references.pmid: 19074352
references.citation: Am J Med. 1977 May;62(5):707-14
references.pmid: 193398
references.citation: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W701-6
references.pmid: 17586823
references.citation: Biochemistry. 2008 Sep 30;47(39):10491-502
references.pmid: 18767813
references.citation: Nature. 2008 Oct 30;455(7217):1251-4
references.pmid: 18820680
references.citation: Nature. 2002 Oct 3;419(6906):520-6
references.pmid: 12368866
references.citation: Mol Cancer. 2009 Sep 28;8:79
references.pmid: 19785722
references.citation: Atherosclerosis. 2003 May;168(1):81-9
references.pmid: 12732390
references.citation: FASEB J. 2008 Dec;22(12):4044-54
references.pmid: 18716026
references.citation: Am J Med. 1951 Oct;11(4):480-93
references.pmid: 14885223
references.citation: Mol Cell Proteomics. 2004 Jul;3(7):729-35
references.pmid: 15102926
references.citation: J Lipid Res. 2009 Apr;50 Suppl:S167-71
references.pmid: 19060251
references.citation: Methods. 2005 Mar;35(3):211-22
references.pmid: 15722218
references.citation: J Lipid Res. 1987 Dec;28(12):1405-9
references.pmid: 3123586
references.citation: J Am Soc Mass Spectrom. 2007 Feb;18(2):226-33
references.pmid: 17070068
references.citation: Anal Chem. 2003 Sep 15;75(18):4818-26
references.pmid: 14674459
references.citation: Biochim Biophys Acta. 2006 Dec;1764(12):1811-22
references.pmid: 17118725
references.citation: Science. 2006 Apr 14;312(5771):212-7
references.pmid: 16614208
references.citation: Lancet. 1975 Jan 4;1(7897):16-9
references.pmid: 46338
references.citation: J Clin Invest. 2007 Dec;117(12):3900-8
references.pmid: 17992262
references.citation: J Proteome Res. 2009 Feb;8(2):603-12
references.pmid: 19055479
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33
references.pmid: 15210983
references.citation: J Proteome Res. 2008 Aug;7(8):3127-36
references.pmid: 18613715
references.citation: Lancet. 1977 May 7;1(8019):965-8
references.pmid: 67464
references.citation: J Proteome Res. 2008 Oct;7(10):4373-83
references.pmid: 18785765
references.citation: FASEB J. 2008 Apr;22(4):1073-82
references.pmid: 18039927