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Affinity Depletion Versus Relative Protein Enrichment

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Affinity Depletion Versus Relative Protein Enrichment Jankovska et al. Clin Proteom (2019) 16:9 https://doi.org/10.1186/s12014-019-9229-1 Clinical Proteomics RESEARCH Open Access Afnity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fuid proteome coverage Eliska Jankovska1, Marek Svitek2,3, Karel Holada4 and Jiri Petrak1* Abstract Cerebrospinal fuid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is compli- cated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple afnity removal systems and protein enrichment of less abundant proteins using a combinatorial pep- tide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome cover- age has never been evaluated side-by-side in a single study. Therefore, we explored the efect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identifed in subsequent LC–MS/MS analysis. LC–MS/MS analysis of crude (non-treated) CSF provided roughly 500 identifed proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifcations to nearly 800, while treatment of CSF using ProteoMiner enabled identifcation of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the “waste” fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the fow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identifed in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workfow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies signifcantly increased the number of identifed CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identifed proteins (773) compared to ProteoM- iner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded “waste” fractions contain proteins of potential interest and should be included in CSF biomarker studies. Keywords: Cerebrospinal fuid, Depletion, CNS, Biomarkers, LC–MS/MS, Mass spectrometry *Correspondence: [email protected] 1 BIOCEV, First Faculty of Medicine, Charles University, Prague, Czech Republic Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jankovska et al. Clin Proteom (2019) 16:9 Page 2 of 10 Introduction removal of the most abundant proteins before LC–MS/ Proteomic analysis of human body fuids is a promising MS is needed to increase the coverage of CSF pro- tool for the identifcation and detection of disease bio- teome [8]. Since there is no specifc method for deal- markers. Human cerebrospinal fuid (CSF) is in direct ing with the abundant proteins in CSF, researchers have contact with the central nervous system (CNS) and to resort to one of the methods developed for blood serves multiple functions including mechanic protection serum/plasma. Afnity depletion of the most abundant of the brain tissue, homeostasis maintenance, delivery of proteins and relative enrichment of medium and low nutrients to the CNS, removal of waste and active regula- abundance proteins using combinatorial peptide ligand tion of CNS via hormones, neuropeptides, and other reg- library are among the most common. ulatory molecules. Te composition of CSF thus refects Afnity depletion is based on the specifc capture of the physiological or pathological status of CNS and target proteins using immobilized antibodies or other makes CSF an attractive source of potential biomarkers molecules (protein A/G, Cibacron Blue) with high afn- for neurologic diseases. ity and specifcity for the targets. Simultaneous removal Abundances of several CSF proteins have been of multiple high abundant proteins has become the approved as diagnostic or prognostic markers in clinical major pre-analytical strategy for proteomic analysis of practice. For instance, total tau protein concentration, blood plasma [9]. Among the most used afnity deple- phospho-tau concentration and presence of 42 amino tion systems belong Multiple Afnity Removal Systems acid form of β-amyloid are indicative of Alzheimer´s dis- (MARS, Agilent) for depleting 2, 6, 7 or 14 most abun- ease [1]. Similarly, the appearance of oligoclonal immu- dant proteins [10, 11], Seppro-IgY-14 (Sigma-Aldrich) noglobulin bands on electrophoretic gels of a CSF sample and ProteoPrep 20 (Sigma-Aldrich) [12, 13] which suggest the presence of multiple sclerosis [2]. However, remove 14 and 20 most abundant plasma proteins, for most diseases of the CNS, there are currently no relia- respectively. ble biomarkers with diagnostic, prognostic or therapeutic An alternative approach to the afnity depletion is signifcance. New biomarkers for neurological diseases the strategy of relative enrichment of low and medium are therefore desirable. abundance proteins based on their interactions with CSF is formed partly (~ 20%) by active secretion of an immobilized combinatorial peptide ligand library, ependymal cells in the choroid plexuses of the brain ven- known under its market name ProteoMiner (Bio-Rad) tricles but mostly by ultrafltration of blood plasma by [14]. Here, proteins from a complex sample bind to a vast choroidal capillaries. CSF composition thus inevitably spectrum of immobilized hexapeptide ligands through refects the composition of blood plasma. However, the various types of interactions (ionic, hydrophobic inter- total protein concentration in CSF is 50–100 times lower, actions, hydrogen bonding, and van der Waal´s force) compared to plasma. While typical plasma protein con- with diferent afnities. Te binding partners for each centration ranges roughly between 60 and 70 mg/ml, protein are present in limited numbers in the library; normal CSF protein concentration is 0.2–0.7 mg/ml [3]. high-abundance proteins thus exceed the concentration Proteomic analysis of CSF is, similarly to blood plasma, of their binders, and their excess copies are washed of. complicated by the complexity of its protein composition, Low-abundance proteins do not saturate their ligands by a very high dynamic range of concentrations of indi- and become relatively concentrated on the beads [15]. vidual proteins and, most importantly, by the presence of Tis approach is expected to enrich medium- and low- several highly abundant proteins [4]. Te ten most abun- abundance proteins while relatively depleting or diluting dant CSF proteins (albumin, IgG, transthyretin, trans- the highly-abundant ones. ferrin, α1-antitrypsin, apolipoprotein A, alpha-1-acid Both strategies, afnity depletion [12, 13] and relative glycoprotein, haptoglobin, α2-macroglobulin, comple- enrichment by a ligand library [16, 17] have been shown ment C3) are the same as in blood plasma and account to signifcantly increase CSF proteome coverage, but their for more than 80% of total CSF protein concentration. relative efect has never been directly compared in a sin- In CSF albumin alone accounts for 60% of total protein gle study. Here, we evaluate the relative beneft of these exceeding its relative abundance in plasma. On the other two pre-analytical strategies side-by-side. Specifcally, we side, the relative abundance of immunoglobulins, namely evaluate the efect of Multiple Afnity Removal System IgG and IgM is signifcantly lower in CSF compared to for depletion of 14 most abundant proteins (MARS 14) plasma [5, 6]. Furthermore, CSF contains specifc high and ProteoMiner ligand library on the number of pro- abundance proteins, for example, cystatin C and prosta- teins identifed in human CSF using a standard LC–MS/ glandin D2 synthase, both being synthesized in CNS [7]. MS setup. Since the ultimate goal of both strategies is rel- High concentrations of the major proteins limit the ative depletion of highly abundant proteins, we will use depth of CSF proteomic analysis and at least partial the term “depletion” for both throughout the manuscript. Jankovska et al. Clin Proteom (2019) 16:9 Page 3 of 10 Materials and methods several steps. After each centrifugation (100×g, 1 min) Materials depleted CSF (fow through) was collected. Te cartridge Acetonitrile (ACN) and ammonium bicarbonate was then washed with 2 × 400 µl of Bufer A. Both washes (AMBIC) were purchased from Fluka (New Jersey, USA), were combined with the depleted CSF and concentrated Dulbecco’s phosphate bufered saline (DPBS), sodium using 5 kDa MWCO
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