JOURNAL OF PROTEOMICS 75 (2011) 211– 220 available at www.sciencedirect.com www.elsevier.com/locate/jprot MRM assay for quantitation of complement components in human blood plasma — a feasibility study on multiple sclerosis Melinda Rezeli a,⁎, Ákos Végvári a, Jan Ottervald b, c, Tomas Olsson b, Thomas Laurell a, d, György Marko-Varga a a Division of Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Lund, Sweden b Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Hospital, Stockholm, Sweden c AstraZeneca, R&D Södertälje, Sweden d Department of Biomedical Engineering, Dongguk University, Seoul, Republic of Korea ARTICLE INFO ABSTRACT Article history: As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for Received 1 March 2011 quantitation of proteotypic peptides, representing seven plasma proteins associated with Accepted 17 May 2011 inflammation (complement components and C-reactive protein). The assay development Available online 15 June 2011 and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable ≤ Keywords: reproducibility over a 2 orders of magnitude linear range (RSD 25%). The assay panel was Selected reaction monitoring utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and Linear ion trap 8 control patients. Multiple sclerosis © 2011 Elsevier B.V. All rights reserved. 1. Introduction biomarkers are available to diagnose disease, to predict disease progression, or to monitor treatment effects. However, several Multiple sclerosis (MS) is a common inflammatory disease of the promising approaches have been proposed [2–6]. central nervous system in young adults. Multiple sclerosis can Quantitation of peptide and protein biomarkers in complex appear in three major forms, as i) relapsing-remitting (RRMS), ii) biological matrices, such as human plasma, is a challenging task primary progressive (PPMS) and iii) secondary progressive [7,8]. In biomarker discovery at the verification and the multiple sclerosis (SPMS). Clinically isolated syndrome (CIS), a validation phase, the determination of the levels of high number single neurological episode caused by demyelization, may be of candidate biomarkers in different body fluids in large sample the first indication of a later evolving multiple sclerosis [1]. sets is crucial in future health care developments. The re- Although, its etiology is still unclear, most likely various genetic quirements are rigorous: as the assay must be sensitive, and environmental factors have impact on the disease devel- accurate, and highly reproducible while the high throughput is opment and progression in individuals. Currently, no validated also an important aspect. Currently, the highly sensitive and Abbreviations: RRMS, relapsing-remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; C3, Complement C3; C4, Complement C4; C5, Complement C5; C9, Complement C9; fB, Complement factor B; fH, Complement factor H; CRP, C-reactive protein. ⁎ Corresponding author at: Div. Clinical Protein Science & Imaging, Dept. of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, SE-221 84 Lund, Sweden. Tel.: +46 46 222 3721; fax: +46 46 222 4527. E-mail address: [email protected] (M. Rezeli). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.05.042 212 JOURNAL OF PROTEOMICS 75 (2011) 211– 220 specific immunoassays (mostly sandwich assays) are common- ly used for verification and validation of new biomarkers [9].The success of the immunoassays based on the highly specific antibodies, which are usually not available for the novel Patient Database candidate proteins, and their development is a very expensive and time-consuming process. A further limitation is the Clinical MS restricted potential to develop multiplex immunoassays that data data in all cases will be quality determined by the antibodies utilized. Mass spectrometry-based targeted approaches, such as Primary Secondary selected reaction monitoring (SRM; or MRM in plural) with the Blood CSF Biobank Biobank use of stable isotope-labeled peptide standards, allow accurate peptide and protein quantitation and provide an alternative Samples for Karolinska Lund Primary Biobank Institute University method to antibody-based approaches [10]. SRM is a targeted MS-based technique, generally performed on triple-quadrupole – (QqQ) instruments. In SRM, the parent ion for the targeted Fig. 1 Overview of the current multiple sclerosis study. analyte is selected in the first quadrupole (Q1), followed by frag- mentation in the collision cell (Q2) whereas the third quadrupole In this paper, we report on a feasibility study conducted on a (Q3) is used to monitor selected specific fragment ions. Coherent multiple sclerosis related sample cohort. The study procedure is precursor and fragment ion pairs are called SRM transitions, and outlined in Fig. 1. We developed an MRM assay for quantitation several such transitions are monitored during a single chro- of plasma proteins associated with inflammation that was matographic run. The two mass filters result in highly specific utilized for the analysis of plasma samples from multiple and sensitive responses for the targeted peptides. MRM assays sclerosis patients with i) relapsing-remitting and ii) secondary provide good sensitivity and quantitation of proteins/peptides in progressive status and iii) from control patients. Seven medium a broad dynamic range (exceeding four orders of magnitude abundant proteins were selected within the concentration [11,12]). Clinical Proteomics Technology Assessment for Cancer range from 1 μg/mL to 1.5 mg/mL in human plasma. network of the National Cancer Institute (NCI-CPTAC) conducted a multilaboratory study to demonstrate that the MRM assays based on standardized protocols can be highly reproducible 2. Materials and methods across 8 laboratories and various instrument platforms [13]. The limit of quantitation (LOQ) in human blood plasma 2.1. Materials without any fractionation is ~1 μg/mL [10], which is insufficient to achieve the requirements for plasma biomarker measure- Water (Chromasolv® Plus for HPLC), formic acid (Reagent ments at the medium- and the low abundant proteins. Different grade≥95%), dithiothreitol and iodoacetamide were purchased strategies have been developed to decrease sample complexity from Sigma-Aldrich (Steinheim, Germany); acetonitrile (Hyper- and simultaneously improve sensitivity of MRM assays. With grade for LC-MS) was from Merck (Darmstadt, Germany). the combination of depletion and strong-cation exchange Sequence grade trypsin was purchased from Promega (Madison, chromatographic (SCX) separation, the LOQ was improved to WI). Light and heavy sequences of the target peptides with 1–10 ng/mL by Keshishian et al. [14]. Whiteaker et al. developed a purity higher than 97% were purchased from Thermo Fischer method, called Stable Isotope Standards with Capture by Anti- Scientific (Ulm, Germany). The C-terminal Arginine or Lysine Peptide Antibodies (SISCAPA), by which they could reach the was labeled with 13Cand15N in the heavy forms, providing an detection limit as low as 1.6 ng/mL and with increased sample increased nominal mass of 10 and 8 Da, respectively. volume they could improve it to tens of pg/mL in multiplex assay [15]. Anti-peptide antibodies were used to capture the target 2.2. Clinical materials peptides and their corresponding stable isotope labeled stan- dards, and the sample processing were automatized, therefore, Human plasma samples were provided by Karolinska University the approach has the ability to achieve high throughput. Hospital, from the Neuro Biobank (Prof. Tomas Olsson). A Most of the proteomic MRM assays are performed on triple secondary biobank was established at Lund University, where quadrupole or hybrid triple quadrupole ion trap instruments all samples were logged into the archive with clinical data [11,16–19]. However, linear ion trap mass spectrometers can associated (Fig. 1). All study enrolment followed the recom- also offer an assay platform for protein quantification, as mendations of the Declaration of Helsinki and the study was reported previously [20–22]. approved by the Ethics Committee of the Karolinska Institute The complement system is part of the innate immune #02-365. Oral and written information was given to the patients system, consisting of a number of circulating and membrane- and confirmed consent in writing was received before inclusion. associated proteins. The complement system provides a defense against pathogens in the human body, as the 2.3. Sample preparation activation of the complement system results in the formation of membrane attack complex (MAC) and the release of The seven most abundant proteins were depleted in the plasma anaphylotoxins. It is also implicated in the pathology of samples by using Plasma 7 Multiple Affinity Removal Spin neurodegenerative disorders [23–25], such as Alzheimer's Cartridge (Agilent Technologies, Santa Clara, CA). The first flow- disease, Parkinson's disease and multiple sclerosis. through fractions were denatured by the addition of 8 M urea; JOURNAL OF PROTEOMICS 75 (2011) 211– 220 213 and reduced with 10 mM dithiothreitol (60 min, 37 °C) and plasma equivalent)