On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins.
Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application.



Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Christopher A
CA
Toth
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States. Electronic address: ZKuklenyik@cdc.gov.
Zsuzsanna
Z
Kuklenyik
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Jeffrey I
JI
Jones
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Bryan A
BA
Parks
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Michael S
MS
Gardner
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
David M
DM
Schieltz
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Jon C
JC
Rees
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Michael L
ML
Andrews
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
Lisa G
LG
McWilliams
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
James L
JL
Pirkle
Centers for Disease Control and Prevention, Division of Laboratory Sciences, Atlanta, GA, United States.
John R
JR
Barr
D001053:Apolipoproteins / Q000032:analysis* / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis / Q000379:methods; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid; D005240:Feasibility Studies; D006801:Humans; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
D001053:Apolipoproteins; D015415:Biomarkers; D001798:Blood Proteins; D014357:Trypsin
Anal Chem. 2009 May 15;81(10):3979-85
19364092
Clin Chem. 1990 Apr;36(4):591-7
2182220
Mayo Clin Proc. 1993 Jul;68(7):657-64
8350639
Prog Lipid Res. 1991;30(2-3):105-38
1823932
Rapid Commun Mass Spectrom. 2007;21(16):2585-93
17639571
Anal Bioanal Chem. 2006 Oct;386(3):614-20
16724223
Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14813-8
21873229
Clin Chem. 1994 Apr;40(4):586-92
8149615
Clin Chem. 2012 Apr;58(4):777-81
22307200
J Lipid Res. 2013 Jul;54(7):1939-48
23620136
Rapid Commun Mass Spectrom. 2012 Jan 30;26(2):101-8
22173797
J Proteome Res. 2015 Feb 6;14(2):928-42
25494833
J Lipid Res. 2001 Sep;42(9):1346-67
11518754
J Biol Chem. 2014 Feb 28;289(9):5596-608
24425874
Arterioscler Thromb Vasc Biol. 2010 Dec;30(12):2528-34
20847305
J Chromatogr A. 2009 Mar 27;1216(13):2695-9
18962647
J Lipid Res. 2007 May;48(5):1190-203
17314277
Curr Cardiol Rep. 2013 Jun;15(6):371
23666884
Protein Sci. 2013 Dec;22(12):1820-5
24115173
Clin Chem. 2010 Dec;56(12):1804-13
20923952
Structure. 2012 May 9;20(5):767-79
22579246
Biochemistry. 2004 Jun 29;43(25):8084-93
15209504
Methods. 2015 Jun 15;81:74-85
25766926
J Proteome Res. 2013 Mar 1;12(3):1067-77
23368288
IUBMB Life. 2014 Sep;66(9):616-23
25328986
Biochim Biophys Acta. 1992 Jun 5;1126(1):105-13
1606170
Atheroscler Suppl. 2006 Aug;7(4):23-7
16822722
Anal Chem. 2013 Sep 3;85(17):8039-45
23937592
J Proteomics. 2014 Sep 23;109:143-61
24972322
Clin Chem Lab Med. 2014 Dec;52(12):1695-727
23940067
J Am Heart Assoc. 2013 May 14;2(3):e000130
23672999
J Biol Chem. 2010 Feb 12;285(7):4652-65
19948731
J Biol Chem. 2011 Apr 8;286(14):12495-508
21292766
Proteomics Clin Appl. 2013 Dec;7(11-12):794-801
23970378
J Lipid Res. 2005 Sep;46(9):1999-2006
15995172
J Biol Chem. 2007 Aug 3;282(31):22249-53
17526499
J Biol Chem. 2013 Nov 22;288(47):33997-34008
24121499
Bioanalysis. 2014;6(19):2685-98
25411709
Biochemistry. 2012 Jun 12;51(23):4633-41
22631438
J Immunol Methods. 2009 Aug 15;347(1-2):3-11
19538965