Journal of Chromatography B, 877 (2009) 1281–1291 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Inﬂuence of clotting time on the protein composition of serum samples based on LC–MS data  Natalia I. Govorukhina a , Marcel de Vries b , Theo H. Reijmers c , Péter Horvatovich a , Ate G.J. van der Zee d , Rainer Bischoff a,∗ a Department of Analytical Biochemistry, Centre for Pharmacy, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands b Mass Spectrometry Core Facility, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands c Division of Analytical Biosciences, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands d Department of Gynecology, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands article info Article history: Received 24 April 2008 Accepted 16 October 2008 Available online 1 November 2008 Keywords: Biomarker Clotting time LC–MS Proteomics Serum abstract Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classiﬁcations especially with respect to long-term follow-up that is otherwise very difﬁcult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may inﬂuence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the inﬂuence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label- free analysis of trypsin-digested serum showed that the overall pattern of LC–MS data is not affected by clotting times varying from 2 to 8 h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived ﬁbrinopeptides, change signiﬁcantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of ﬁbrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC–MS data using serum sample collections of which the clotting time is not known. Next to the ﬁbrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1h. For prospective studies, we recommend to let blood clot for at least 2h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The discovery and validation of biomarkers for early diagnosis of disease at a stage where successful therapy is still possible is an important goal of modern biomedical research. To achieve this goal, high-resolution analytical techniques are applied to complex clinical samples, mostly body ﬂuids. Serum is a body ﬂuid that is representative of the composition of soluble proteins and peptides in blood and is thus a suitable starting material for biomarker dis- covery studies. Moreover, many existing large sample collections at major hospitals consist of serum that is stored frozen at -80 ◦ C. Since these collections may well contain important information  This paper is part of the special issue “Quantitative Analysis of Biomarkers by LC–MS/MS”, J. Cummings, R.D. Unwin and T. Veenstra (Guest Editors). ∗ Corresponding author. Tel.: +31 50 363 3338; fax: +31 50 363 7582. E-mail address: r.p.h.bischoff@rug.nl (R. Bischoff). about the health status of the corresponding patients and controls, especially when followed over long periods of time, it is critical to evaluate under which conditions it is possible to compare samples from these collections with modern proteomics approaches. The generation of serum requires that blood be coagulated and that the cellular components as well as the blood clot be removed by centrifugation or ﬁltration. It has notably been argued that the time and conditions under which blood is allowed to clot (clot- ting time) are important parameters that must be controlled and kept constant in order to compare protein and peptide proﬁles [1–5]. However, most existing sample collections have not been obtained with subsequent proteomics analyses in mind and clot- ting time and conditions have often not been rigorously controlled. Many of the studies evaluating the inﬂuence of pre-analytical parameters on serum protein composition have been performed by surface-enhanced laser desorption ionization time-of-ﬂight mass spectrometry (SELDI-TOF-MS) [4], a method that suffers from rather poor concentration sensitivity and that may be prone to 1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.10.029