Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC‐MS/MS. Issue 11 (31st October 2013)

• Record Type: Journal Article
• Authors: Ceglarek, Uta; Dittrich, Julia; Becker, Susen; Baumann, Frank; Kortz, Linda; Thiery, Joachim; Sauer, Sascha
• Is Part Of: Proteomics. Volume 7:Issue 11/12(2013)
• Page Start: 794
• Page End: 801
• Publication Date: 2013-10-31
• Subjects: Proteomics -- Periodicals; 572.605
• DOI: 10.1002/prca.201300034
• Languages: English
• ISSNs: 1862-8346

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="prca1491-sec-0010" sec-type="section"> <title>Purpose</title> <p>We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of seven apolipoproteins (Apos) in human serum by LC‐MS/MS using proteotypic peptides and corresponding stable isotope‐labeled peptides as internal standards.</p> </sec> <sec id="prca1491-sec-0020" sec-type="section"> <title>Experimental design</title> <p>Micro‐LC was coupled with quadrupole‐linear ion trap MS for quantification and peptide confirmation. Denaturation, reduction, alkylation, and tryptic digestion including ultrasound and microwave assistance were investigated. Method comparison of 50 plasma samples with an immunoassay was performed for Apo A‐I and Apo B.</p> </sec> <sec id="prca1491-sec-0030" sec-type="section"> <title>Results</title> <p>Tryptic digestion times ranged between 5 min (Apo A‐I, Apo E, Apo A‐IV) and 16 h (Apo A‐II). Ultrasound and microwave assistance did not improve the digestion yield. Linearity was found between 0.1 nmol/L and 100 mmol/L. The lower limits of quantification were ≤0.4 μmol/L for Apo A‐I, Apo A‐IV, Apo B‐100, Apo C‐I, Apo C‐III, Apo E, and <1.4 μmol/L for Apo A‐II. CV <13% were determined. Comparison with immunoassays showed a good agreement for Apo A‐I and Apo B.</p> </sec> <sec id="prca1491-sec-0040" sec-type="section"> <title>Conclusion and clinical relevance</title> <p>The validated preanalytical protocol enables a reliable simultaneous analysis of seven Apos in human serum without depletion. The method can now be applied in clinical studies to investigate the Apo distributions in cardiovascular diseases.</p> </sec> </abstract>