Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry.
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.
Mazur
Matthew T
MT
Department of Proteomics, Merck Research Labs, 126 E Lincoln Avenue, PO Box 2000, Rahway, NJ 07065, USA.
Cardasis
Helene L
HL
Spellman
Daniel S
DS
Liaw
Andy
A
Yates
Nathan A
NA
Hendrickson
Ronald C
RC
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*
D053305:Apolipoprotein C-III; D008076:Cholesterol, HDL; D020033:Protein Isoforms
Proteomics. 2006 Apr;6(7):2101-7
16518871
Science. 1999 May 21;284(5418):1289-90
10383309
Annu Rev Biochem. 2001;70:437-73
11395414
Anal Chem. 2004 Oct 15;76(20):6085-96
15481957
Nat Methods. 2007 Oct;4(10):817-21
17901871
Ann Med. 2009;41(1):11-8
19085339
Anal Biochem. 1985 Oct;150(1):76-85
3843705
Nature. 2007 Dec 13;450(7172):991-1000
18075578
J Proteome Res. 2010 Mar 5;9(3):1392-401
20095649
Int J Mass Spectrom. 2007 Jan 1;259(1-3):197-203
17364019
Proc Natl Acad Sci U S A. 1965 Dec;54(6):1699-705
5218921
J Clin Invest. 2007 Mar;117(3):746-56
17332893
J Lipid Res. 1981 Feb;22(2):339-58
6787159
Science. 2008 Dec 12;322(5908):1702-5
19074352
Am J Med. 1977 May;62(5):707-14
193398
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W701-6
17586823
Biochemistry. 2008 Sep 30;47(39):10491-502
18767813
Nature. 2008 Oct 30;455(7217):1251-4
18820680
Nature. 2002 Oct 3;419(6906):520-6
12368866
Mol Cancer. 2009 Sep 28;8:79
19785722
Atherosclerosis. 2003 May;168(1):81-9
12732390
FASEB J. 2008 Dec;22(12):4044-54
18716026
Am J Med. 1951 Oct;11(4):480-93
14885223
Mol Cell Proteomics. 2004 Jul;3(7):729-35
15102926
J Lipid Res. 2009 Apr;50 Suppl:S167-71
19060251
Methods. 2005 Mar;35(3):211-22
15722218
J Lipid Res. 1987 Dec;28(12):1405-9
3123586
J Am Soc Mass Spectrom. 2007 Feb;18(2):226-33
17070068
Anal Chem. 2003 Sep 15;75(18):4818-26
14674459
Biochim Biophys Acta. 2006 Dec;1764(12):1811-22
17118725
Science. 2006 Apr 14;312(5771):212-7
16614208
Lancet. 1975 Jan 4;1(7897):16-9
46338
J Clin Invest. 2007 Dec;117(12):3900-8
17992262
J Proteome Res. 2009 Feb;8(2):603-12
19055479
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33
15210983
J Proteome Res. 2008 Aug;7(8):3127-36
18613715
Lancet. 1977 May 7;1(8019):965-8
67464
J Proteome Res. 2008 Oct;7(10):4373-83
18785765
FASEB J. 2008 Apr;22(4):1073-82
18039927