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 spin concentrator to 200 µl. Proteins chloride, trifuoroacetic acid (TFA) and urea were from bound to the cartridge were eluted by 2.5 ml of Bufer Sigma-Aldrich (St. Louis, USA), Bradford protein assay B as the “waste” fraction and stored at − 80 °C until and iodoacetamide (IAA) were from Bio-Rad (Hercules, analysis. USA), dithiothreitol (DTT) and sequencing grade modi- fed trypsin were purchased from Promega (Fitchburg, Relative protein enrichment using ProteoMiner ligand USA). Centrifugal flters 30 kDa (spin concentrators) library and water (Milli-Q) for all experiments were from Merck Protein enrichment was performed using Protein enrich- Millipore (Burlington, USA). Opti-Trap, desalting car- ment small-capacity kit (Bio-Rad, CA, USA) according tridge (C18) was purchased from Optimize Technologies to the manufacturer’s instructions appropriately adapted (Oregon City, USA). Cellulose acetate flters 0.22 µm and to the low concentration of CSF. Te kit is designed for 5 kDa MWCO (molecular weight cut-of) spin concen- 10 mg of proteins. As our CSF samples contained 500 µg trator were purchased from Agilent Technologies (Santa of proteins, we used 25 µl aliquots of beads for each CSF Clara, USA). replicate. Beads were centrifuged (1000×g, 1 min, room temperature) to remove the storage liquid and then CSF collection washed twice with 200 µl of wash bufer. CSF sample Te CSF samples were collected by lumbar puncture containing 500 µg of proteins was added to the beads and during spinal anesthetics procedures in patients of the incubated for 2 h at room temperature in a 3D rotation Urology Clinic of General University Hospital undergo- mixer (RH-18, Hangzhou Miu Instruments). Te sample ing minor surgery. Te study was approved by the Eth- was then centrifuged (1000×g, 1 min, room temperature) ics Committee of General University Hospital, Prague. for 1 min, and the fow-through fraction containing the CSF samples were centrifuged at 1500×g for 10 min at unbound waste was collected. Te beads with enriched 4 °C within 30 min to remove cellular debris. Samples proteins were washed twice with 200 µl of wash bufer were then ultracentrifuged (Beckman Coulter Optima and once with 200 µl of MiliQ water after 5 min incu- LE-80 K Ultracentrifuge, rotor SW 28, 120,000×g, 2 h, bation on the 3D rotation mixer, all three washes were 4 °C) to remove all membranous vesicles and other parti- joined with the waste fraction. Bound proteins were cles. Obtained supernatants were aliquoted and stored at eluted from the beads in three steps each consisting of − 80 °C until analysis. 15 min incubation in 20 µl of elution reagent and centrif- ugation. Te three eluate samples were combined repre- Preparation of pooled CSF sample senting the depleted CSF. Individual CSF samples were thawed on ice and protein concentration was determined using Bradford protein Reduction, alkylation, digestion, and desalting assay (Bio-Rad) at 595 nm. A pooled CSF sample was Triplicates of CSF samples processed by both workfows generated from 5 patient samples. Each patient’s sample and triplicates of crude CSF (representing 100 µg of total contributed to the pool the same amount of protein. Te protein each) were transferred to 30 kDa cut of flters. pooled CSF sample was divided into aliquots, each repre- Te subsequent reduction, alkylation, and digestion senting 500 µg of total protein. Tese aliquots were used of the samples were performed using the FASP (flter- throughout the study, always in technical triplicates. aided sample preparation) protocol [18]. Samples were alkylated for 20 min at room temperature in the dark by Multiple afnity depletion using MARS 14 cartridge 100 µl of iodoacetamide solution (50 mM iodoacetamide Samples were processed in triplicates, according to in 8 M urea, 0.1 M Tris/HCl pH 8.5). Samples were then manufacturer´s instructions. MARS 14 (Agilent Technol- washed twice with 50 mM ammonium bicarbonate, and ogies, Santa Clara, USA) cartridge was tempered at room proteomics grade trypsin was added. Te samples were temperature for 20 min. Ten the cartridge was equili- digested overnight at 37 °C and peptides were collected brated with 4 ml of Bufer A. CSF sample (500 µg) was by centrifugation. Te peptides were desalted on Opti- diluted 1:1 in Bufer A and was fltered through 0.22 µm Prep C-18 manually operated cartridge. Te cartridge flter (10,000×g, 10 min, 4 °C). Since the volume capac- was equilibrated by 700 µl of 80% ACN in 0.1% TFA fol- ity of the spin cartridge is limited the diluted CSF sam- lowed by 600 µl of 0.1% TFA. Te sample was loaded on ple was loaded on the MARS 14 cartridge sequentially in the desalting cartridge and washed with 500 µl of 0.1% Jankovska et al. Clin Proteom (2019) 16:9 Page 4 of 10

TFA. Peptides were eluted by 200 µl of 80% ACN in 0.1% 0.7 min). Te “match between runs” was also used in TFA, dried in a speedvac (Eppendorf, Concentrator Plus) quantifcation experiments. Quantifcations were per- and stored at − 80 °C. formed with the label-free algorithm in MaxQuant. Data analysis was performed using Perseus 1.6.1.3. nLC–MS2 analysis software. LC–MS/MS analyses were performed on a Termo Orbitrap Fusion (Q-OT-qIT, Termo) mass spectrometer equipped with Dionex Ultimate 3000 chromatograph. Data presentation Samples were loaded onto the trap column (Acclaim Pep- All Venn diagrams were created by the web application BioVenn [19]. Map300, C18, 5 µm, 300 Å Wide Pore, 300 µm × 5 mm, 5 Cartridges) for 4 min at 15 μl/min in 2% acetonitrile in 0.1% TFA. EASY-Spray column, 50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 Å pore size was used Results and discussion for the separation. Mobile phase A was composed of We evaluated the efect of the two major methods for 2% acetonitrile in 0.1% formic acid. Mobile phase B was depletion of abundant plasma proteins in CSF. Specif- composed of 80% acetonitrile in 0.1% formic acid. Te cally, we assessed the impact of MARS 14 cartridge and gradient was 90 min long at fow-rate 300 nl/min and ProteoMiner immobilized library on the number of temperature 55 °C. Mobile phase B increases from 2 to proteins identifed in triplicate CSF samples by a stand- 40% B at 60 min, 90% B at 61 min, hold for 8 min, and ard LC–MS/MS. In addition to the key fractions of 2% B at 70 min, hold for 15 min until the end of the run. interest, i.e., depleted CSF samples, we also determined Eluting peptide cations were converted to gas-phase ions the number of proteins present in the “waste” fractions, by electrospray ionization and analyzed on a Termo which are routinely discarded. (i.e., proteins retained Orbitrap Fusion. Survey scans of peptide precursors from on MARS 14 cartridge and proteins excluded by the 400 to 1600 m/z were performed at 120 K resolution (at ligand library and contained in the ProteoMiner fow- 5 through fraction). All samples originated from a single 200 m/z) with a 5 × 10 ion count target. Tandem MS was performed by isolation at 1,5 T with the quadrupole, CSF sample pooled from 5 patients. A non-fractionated HCD fragmentation with a normalized collision energy crude CSF sample was used as a reference; all samples of 30, and rapid scan MS analysis in the ion trap. Te were processed in technical triplicates. Te experimen- MS2 ion count target was set to 10­ 4 and the max injec- tal workfow is depicted in Fig. 1. tion time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS2. Te dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. Te instrument was run in top speed mode with 2 s cycles.

Data analysis Te raw data obtained by LC–MS/MS were analyzed using MaxQuant software (version 1.6.0.1). Te false discovery rate (FDR) was set to 1% for both, proteins and peptides. Te minimum peptide length was set to 7 amino acids. Te enzyme was set to trypsin with a maxi- mum of two missed cleavages. Carbamidomethylation of cysteines was set as a fxed modifcation. N-terminal protein acetylation and methionine oxidation as variable modifcations. Main search peptide tolerance was set to 4.5 ppm. MS/MS mass tolerance was set to 0.5 Da. Te Andromeda search engine was used for the MS/ MS spectra search against the UniProt Human data- base (downloaded in March 2018). Te “match between runs” feature of MaxQuant was used to transfer iden- Fig. 1 Experimental workfow. Aliquots of a pooled human CSF tifcations to other LC–MS/MS runs based on their sample were used. All analyses were performed in triplicates masses and retention time (maximum deviation of Jankovska et al. Clin Proteom (2019) 16:9 Page 5 of 10

All proteins identifed in the crude CSF, depleted CSF ProteoMiner hexapeptide ligand library increased the and corresponding waste fractions are listed in Addi- number of identifed CSF proteins by 29% compared to tional fle 1. crude CSF. Te total number of unique proteins identi- fed in the ProteoMiner-treated CSF triplicate was 611 Crude CSF (Fig. 2), 366 proteins were identifed across all three LC–MS/MS analysis of crude CSF triplicates provided replicates. 397, 419 and 427 identifed proteins (Fig. 2). When the Considering the number of identifed proteins, it is evi- MS data of the triplicates were searched together, the dent, that both depletion strategies are efective and sig- analysis resulted in the identifcation of a total of 475 nifcantly increase the number of identifed proteins in unique proteins, 275 proteins were present in all three comparison to crude CSF. However, MARS 14 immuno- replicates. depletion enabled identifcation of a substantially more Te number of identifed proteins is with an agreement proteins. with a previous study using comparable LC–MS/MS setup [20]. Te proteome coverage obtained for crude Depletion efciency CSF was used as a reference for evaluation of the efect of To evaluate the efciency of depletion of the most abun- CSF processing with MARS 14 cartridge and ProteoM- dant proteins we used label-free quantitative (LFQ) iner library. analysis. Relative depletion of the major proteins was calculated from the normalized intensities of peptide sig- CSF depleted by MARS 14 cartridge nals in the crude CSF and the depleted CSF. Results can To evaluate the beneft of immunodepletion of major be seen in Table 1. Te depletion efciency of MARS 14 plasma proteins we used Multiple afnity removal cartridge for the 14 most abundant (plasma) proteins was cartridge for depletion of the 14 most abundant pro- high, comparable with the efciency claimed by the man- teins of human plasma (MARS 14, Agilent), very often ufacturer and confrmed by others [10]. In general, CSF used in proteomic biomarker studies. Depletion of the depletion with ProteoMiner decreased the amounts of most abundant CSF proteins using MARS 14 cartridge the individual 14 most abundant proteins less efectively. increased the number of identifed proteins by 63% com- pared to crude CSF to total 773 in the triplicate analysis The depleted CSF (Fig. 2). We identifed between 633 and 696 proteins in Te two compared strategies employ radically difer- the triplicate analyses. Of the total 773 identifed pro- ent principles of dealing with the most abundant pro- teins, 480 proteins were found in all three replicates. teins, i.e., specifc interactions with antibodies and other molecules versus less defned interactions with a library CSF depleted with ProteoMiner ligand library of peptide ligands. Also, MARS 14 cartridge retains Te principally diferent method of relative protein unwanted proteins bound on the stationary phase, while enrichment of medium and low abundance protein using

Fig. 2 CSF proteome coverage. Numbers of proteins identifed by LC–MS/MS in the triplicate analyses of crude CSF, CSF depleted by MARS 14 and CSF depleted by ProteoMiner Jankovska et al. Clin Proteom (2019) 16:9 Page 6 of 10

Table 1 The efciency of depletion of the most abundant between the two depleted fractions neither between the 14 plasma proteins by the MARS 14 immunodepletion depleted samples and crude CSF (Additional fle 2). and ProteoMiner One example of depletion bias between these two MARS 14 ProteoMiner methods is a higher number of various immunoglobulins %Removed %Removed found in the CSF sample depleted by ProteoMiner (33 identifed proteins) compared to CSF treated by MARS P02768 Serum albumin ALB 97.4 71.9 14 (10 proteins). In this case, it can be easily explained P02647 Apolipoprotein A-I APOA1 99.9 < 33 by the fact that MARS 14 specifcally and efciently tar- P02652 Apolipoprotein A-II APOA2 75.6 < 33 gets and depletes immunoglobulins. However, among P01876 Ig alpha-1 chain C IGHA1 99.9 97.8 region the method-specifc proteins were also molecules previ- P01023 Alpha-2-macroglob- A2M 99.9 68.4 ously identifed as specifc brain-enriched proteins [21] ulin and therefore molecules with biomarker potential. For P02763 Alpha-1-acid glyco- ORM1 99.7 97.8 example, brain-enriched delta and Notch-like epidermal protein 1 growth factor-related receptor (DNER_HUMAN), or P01009 Alpha-1-antitrypsin SERPINA1 99.7 84.3 Myelin-associated glycoprotein (MAG_HUMAN) were P01857 Ig gamma-1 chain C IGHG1 99.6 70.9 present only in the CSF depleted by MARS 14 while Pro- region tocadherin-8 (PCDH8_HUMAN) and Protocadherin P02787 Serotransferrin TF 99.4 96.1 gamma-C5 (PCDGM_HUMAN) were identifed only P00738 Haptoglobin HP 98.7 92.1 in the CSF sample after ProteoMiner depletion [21]. P01024 Complement C3 C3 98.1 <33 In summary, both depletion methods have a profound P02766 Transthyretin TTR​ 95.2 89.0 benefcial impact on CSF proteome coverage. However, P02675 Fibrinogen beta chain FGB 94.5 < 33 the lists of identifed proteins difer between the two P02671 Fibrinogen alpha FGA 93.7 < 33 chain approaches suggesting a potential loss of biomarker pro- P02679 Fibrinogen gamma FGG 91.7 < 33 teins. To further explore the issue we analyzed the waste chain fractions generated in both workfows. P01871 IgM IGHM 100a < 33

Data from label-free quantifcation based on MS1 peptide signal intensities The waste fractions a IgM was not detected in the CSF after MARS 14 immunodepletion Several studies reported that immunodepletion of high- abundant proteins leads to an undesired co-depletion of numerous non-targeted proteins from the CSF [12, 13], ProteoMiner excludes the abundant proteins in the including potential biomarkers of neurologic diseases mobile phase. [22]. Although we found no comparable study addressing Te principal diferences in the two depletion strate- the losses of CSF proteins in the ProteoMiner workfow, gies and their diferent depletion efciencies may afect it can be expected that analogical losses occur, either due not only the number of identifed CSF proteins but can to low binding to the hexapeptide library or due to strong also result in the identifcation of diferent sets of pro- interactions with the abundant proteins that oversaturate teins. We compared the lists of proteins identifed in CSF their binding ligands. depleted by MARS 14 and ProteoMiner and confrmed To compare the extent of such unwanted protein losses that both methods provided a large number of unique due to co-depletion in both workfows, we analyzed the proteins not identifed by the other method (Fig. 3). Te fraction of proteins retained by the MARS 14 cartridge depletion of CSF using MARS 14 cartridge provided 336 and the equivalent proteins present in the fow-through such proteins not identifed in the CSF treated by Prot- fraction of the ProteoMiner library workfow. Tese eoMiner and vice versa, the CSF sample treated with Pro- waste fractions are routinely excluded from further anal- teoMiner provided 174 unique proteins not observed in ysis and discarded. the CSF depleted by MARS 14. Tis suggests that both LC–MS/MS analysis of the waste fractions revealed methods introduce a bias toward and against some par- signifcant extent of CSF protein loss in both deple- ticular proteins. tion workfows. In addition to the most abundant CSF We evaluated three basic properties of the identifed proteins (albumin, IgG, transthyretin, transferrin, α-1- proteins, namely MW, pI and hydrophobicity (GRAVY antitrypsin, haptoglobin α, etc.), both waste fractions score) of all proteins identifed in the depleted CSF sam- contained a substantial number of co-depleted non-tar- ples. Tere was no systematic preference regarding MW, get proteins. MARS 14 cartridge retained 214 proteins, pI and hydrophobicity (GRAVY score) distributions and the ProteoMiner fow-through fraction contained 272 identifed proteins (Fig. 4a). Jankovska et al. Clin Proteom (2019) 16:9 Page 7 of 10

in the ProteoMiner waste (91% identifed proteins), while in the MARS 14 waste only 64% of identifed proteins were also seen in the crude CSF (Fig. 4a). Although the numbers of the proteins identifed in both waste frac- tions were roughly comparable (214 vs. 272), the lists of the proteins difered signifcantly, only 111 proteins were present in both fractions. 103 and 161 proteins were spe- cifc for MARS 14 and ProteoMiner waste, respectively (Fig. 4b). Both these observations seem to refect the fundamentally diferent fractionation principles of the depletion methods. However, again there was no system- atic bias regarding MW, pI and hydrophobicity (GRAVY score) distributions between the two waste fractions or compared to depleted or crude CSF samples (see Addi- tional fle 2). Te high numbers of proteins identifed in the waste Fig. 3 The unique and shared proteins identifed in depleted CSF fractions confrm the alarmingly large extent of the after depletion by MARS 14 or ProteoMiner. Over 330 proteins unwanted loss of proteins in both workfows. Impor- identifed in CSF depleted by MARS 14 cartridge were not found in tantly, many of the CSF proteins identifed in the waste the CSF depleted by ProteoMiner library. Vice versa, 174 proteins fractions were unique for the waste, i.e., were not found were uniquely identifed only in the CSF sample after ProteoMiner depletion in the corresponding depleted CSF samples. We identi- fed 115 and 69 such proteins in MARS 14 and Prot- eoMiner waste fractions, respectively (Fig. 5). Addition of the waste-specifc proteins to the list of proteins Te signifcant number of the proteins identifed in the identifed in the corresponding depleted CSF increased waste fractions was also identifed during the analysis of the total coverage of CSF proteome roughly by 10-15 crude CSF. Large overlap with the crude CSF was found percent to 888 and 680 identifed proteins in MARS

Fig. 4 Proteins identifed in the waste fractions (proteins retained by MARS 14 cartridge and proteins excluded in the ProteoMiner fow-through). a The overlap of the proteins identifed in the waste fractions with the proteins found in crude CSF. b The overlap between the two waste fractions Jankovska et al. Clin Proteom (2019) 16:9 Page 8 of 10

Fig. 5 The total number of proteins identifed in the depleted CSF and corresponding waste fractions

14 and ProteoMiner workfow, respectively (Fig. 5). as a potential marker of dementia and acute neuroin- Te strikingly high number of proteins lost during fammatory disorders in the form of its stable frag- both depletion workfows may advocate future inclu- ment (MR-PENK A) [25]. Proenkephalin-A was also sion of the waste fractions into CSF biomarker stud- described as an indicator of severity and clinical out- ies. Such inclusion would be benefcial, especially if come in patients with ischemic stroke [26]. the waste fraction contained not only the major plasma constituents but also CNS-specifc proteins. To evalu- Conclusions ate the presence of such molecules, we compared the CSF is a valuable source of information on the status of lists of proteins identifed in the waste fractions with CNS and a potential treasure trove of biomarkers. Com- a list of previously characterized as brain-enriched pared to blood plasma, proteomic analysis of CSF is lim- proteins [21]. We found several such molecules to be ited by its low protein concentration and the maximum present in the waste fractions, namely cell adhesion volume of CSF that can be safely taken during the lumbar molecule 2 (CADM2_HUMAN), Amyloid-like pro- puncture. Considering also the potential health risks of tein 1 (APLP1_HUMAN) or Beta-Ala-His dipeptidase the lumbar puncture, it is imperative to make proteomic (CNDP1_HUMAN). Further search in literature iden- analyses of CSF as efcient as possible. To maximize the tifed additional waste-specifc proteins with known number of identifed CSF proteins we compared the ben- relevance for neurophysiology and neuropathology. For efts of two frequent, but principally diferent, methods example, MARS 14 cartridge fully retained Kinesin- for relative depletion of major plasma proteins—MARS like protein KIF1B (KIF1B_HUMAN) linked to Mul- 14 cartridge and ProteoMiner ligand library. To our tiple Sclerosis susceptibility [23] and Tubulin beta-3 knowledge, this is the frst side-by-side comparison of chain (TBB3_HUMAN) required for axon guidance these two major strategies in CSF. In our hands, both [24]. Similarly, ProteoMiner waste fraction (but not methods markedly improved CSF proteome coverage CSF depleted by ProteoMiner) contained Proenkepha- compared to analysis of crude (non-depleted) CSF. How- lin-A (PENK_HUMAN) which has been considered ever, both methods enabled identifcation of exclusive Jankovska et al. Clin Proteom (2019) 16:9 Page 9 of 10

sets of proteins in the resulting depleted CSF, refecting communication [27] and can be isolated from patient the distinct principles of the fractionation. CSF and analyzed by LC–MS/MS [28]. Similarly, endog- Te obvious benefcial efect of major protein deple- enous (neuro)peptides can be released from their carrier tion, however, comes for the price of unwanted protein proteins, collected by ultrafltration and analyzed [29]. loss during the procedure. Depletion or removal of any Such semi-targeted approaches may reveal a brand new protein from a complex protein mixture under non- level of information carried by CSF. denaturing conditions inevitably leads to a co-depletion of other, non-targeted, proteins. In the case of MARS 14, it is either due to their direct interaction with the tar- Additional fles geted proteins, due to cross-reactivity or a nonspecifc interaction with the antibody, or via interactions with Additional fle 1. A list of all identifed proteins including crude CSF, the stationary phase matrix. In the case of ProteoMiner, depleted CSF and the waste fractions. it may be due to insufcient interaction of proteins with Additional fle 2. Distributions of MW, pI and hydrophobicity (GRAVY score) in crude CSF, depleted CSF and the waste fractions. the hexapeptide library or due to strong interactions with the excluded highly abundant proteins. In both tested Abbreviations methods, we observed a signifcant number of unique ACN: acetonitrile; AMBIC: ammonium bicarbonate; CNS: central nervous co-depleted proteins (proteins not identifed in the cor- system; CSF: cerebrospinal fuid; DTT: dithiothreitol; IAA: iodoacetamide; LC– responding depleted CSF samples), including brain- MS/MS: liquid chromatography—tandem mass spectrometry; MARS: multiple afnity removal system; MW: molecular weight; pI: isoelectric point; TFA: enriched proteins, proteins with potential or confrmed trifuoracetic acid. roles in CNS pathologies—i.e., potential biomarkers. Tis clearly showed, that to maximize CSF proteome coverage Authors’ contributions All authors read and approved the fnal version of the manuscript. EJ and to limit the potential loss of biomarkers during the performed the CSF sample processing, LC–MS/MS analyses and wrote the depletion, proteins retained by the MARS 14 cartridge manuscript. MS collected the CSF samples during spinal anaesthesia. KH (or excluded by ProteoMiner beads) should also be ana- conceived the study and interpreted the data. JP (the corresponding author) analysed and interpreted the data and wrote the manuscript. All authors read lyzed in future CSF biomarker studies. Alternatively, the and approved the fnal manuscript. addition of a crude CSF sample analysis may partially avoid the loss of some proteins due to co-depletion as Author details 1 BIOCEV, First Faculty of Medicine, Charles University, Prague, Czech Republic. the proteins contained in the waste fractions are largely 2 Department of Anesthesiology and Intensive Care, First Faculty of Medicine, a subset of crude CSF. However, in contrast to the waste Charles University, Prague, Czech Republic. 3 General University Hospital, fraction, the inclusion of crude CSF would require an Prague, Czech Republic. 4 Institute of Immunology and Microbiology, First additional volume of CSF. Faculty of Medicine, Charles University, Prague, Czech Republic. Nevertheless, our main aim was to determine which of Acknowledgements the two depletion methods enables higher coverage of the The study was supported by the Ministry of Health of the Czech Republic (NV 15-32961A), by the Ministry of Education, Youth and Sports of the Czech CSF proteome in an identical LC–MS/MS setup. MARS Republic (Progress Q26, NPU II—LQ1604, SVV 260 374, UNCE/MED/016). 14 cartridge enabled markedly higher proteome coverage Authors also acknowledge support by the projects CZ.1.05/2.1.00/19.0400 and with 773 identifed proteins compared to ProteoMiner CZ.1.05/1.1.00/02.0109 from the Research and Development for Innovations Operational Program (RDIOP) co-fnanced by European regional development (611) and crude CSF (475). Te superior performance fund and the state budget of the Czech Republic. of the MARS 14 cartridge can be further emphasized by quicker sample processing. Additionally, depletion Competing interests of a single CSF sample using MARS 14 cartridge is less The authors declare that they have no competing interests. expensive, despite higher acquisition cost of MARS 14 Availability of data and materials cartridge. The dataset supporting the conclusions of this article (the complete list of proteins identifed in the separate CSF fractions including accession numbers To further increase the CSF coverage and make bio- and sequence coverages) is included within the article as “Additional fle 1”. marker discovery more successful, novel strategies may Raw MS data are available from the corresponding author upon request. be needed (in addition to a more extensive peptide frac- Consent for publication tionation). Among such tactics may be an orientation to Not applicable. specifc forms of information carried by the CSF, namely neuropeptides or extracellular vesicles. Tese potential Ethics approval and consent to participate The study was approved by the Ethics Committee of General University Hospi- information carriers are usually excluded or lost in the tal, Prague, Czech Republic. (č.j. 120/14 Grant VES 2015 AZV 1. LF UK). standard proteomic analyses of CSF because of their low MW or sedimentation, respectively. Extracellular Funding The study was supported by the Ministry of Health of the Czech Republic vesicles are cell-derived membrane structures carrying (NV 15-32961A), by the Ministry of Education, Youth and Sports of the Czech tissue-specifc information and play a role in intercellular Republic (Progress Q26, NPU II—LQ1604, SVV 260 374, UNCE/MED/016). Jankovska et al. Clin Proteom (2019) 16:9 Page 10 of 10

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