Combinatorial Peptide Ligand Libraries Research Articles

Pillaging plucking plundering ransacking proteomes via CPLL technology

No proteome can be considered “democratic”, but rather “oligarchic” since a few proteins dominate the landscape and often obliterate the signal of the rare ones. That is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is repeatedly seen. Current pre-fractionation techniques, one way or another, are besieged by problems, in that they are based on a “depletion principle”, i.e. elimination of unwanted species. Yet “democracy” calls for giving “equal rights” to everyone. One way to achieve that would be the use of libraries of combinatorial ligands coupled to spherical beads. When these beads are contacted with complex proteomes (e.g., human urines and sera, egg white, any cell or tissue lysate) of widely differing protein composition and relative abundances, they are able to “normalize” the protein population, by sharply reducing the concentration of the most abundant components while simultaneously enhancing the level of the most dilute components. It is felt that this method could offer a strong step forward in bringing the “unseen proteome” (due to either low abundance and/or presence of interferences) within the detection capabilities of current proteomics detection methods. Examples are given of the normalization of human urine and sera samples, resulting in the discovery of a host of proteins previously unreported. These beads can also be used to remove host cell proteins from purified recombinant proteins or proteins purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with Combinatorial Peptide Ligand Libraries (CPLL), even these impurities can be effectively removed with minute losses of the main, valuable product.

Weighted Gene Co-Expression Network Analysis and Support Vector Machine Learning in the Proteomic Profiling of Cerebrospinal Fluid from Extraventricular Drainage in Child Medulloblastoma.

Medulloblastoma (MB) is the most common pediatric malignant central nervous system tumor. Overall survival in MB depends on treatment tuning. There is aneed for biomarkers of residual disease and recurrence. We analyzed the proteome of waste cerebrospinal fluid (CSF) from extraventricular drainage (EVD) from six children bearing various subtypes of MB and six controls needing EVD insertion for unrelated causes. Samples included total CSF, microvesicles, exosomes, and proteins captured by combinatorial peptide ligand library (CPLL). Liquid chromatography-coupled tandem mass spectrometry proteomics identified 3560 proteins in CSF from control and MB patients, 2412 (67.7%) of which were overlapping, and 346 (9.7%) and 805 (22.6%) were exclusive. Multidimensional scaling analysis discriminated samples. The weighted gene co-expression network analysis (WGCNA) identified those modules functionally associated with the samples. A ranked core of 192 proteins allowed distinguishing between control and MB samples. Machine learning highlighted long-chain fatty acid transport protein 4 (SLC27A4) and laminin B-type (LMNB1) as proteins that maximized the discrimination between control and MB samples. Machine learning WGCNA and support vector machine learning were able to distinguish between MB versus non-tumor/hemorrhagic controls. The two potential protein biomarkers for the discrimination between control and MB may guide therapy and predict recurrences, improving the MB patients’ quality of life.

Comprehensive Plasma N-Glycoproteome Profiling Based on EThcD-sceHCD-MS/MS.

Glycoproteins are involved in a variety of biological processes. More than one-third of the plasma protein biomarkers of tumors approved by the FDA are glycoproteins, and could improve the diagnostic specificity and/or sensitivity. Therefore, it is of great significance to perform the systematic characterization of plasma N-glycoproteome. In previous studies, we developed an integrated method based on the combinatorial peptide ligand library (CPLL) and stepped collision energy/higher energy collisional dissociation (sceHCD) for comprehensive plasma N-glycoproteome profiling. Recently, we presented a new fragmentation method, EThcD-sceHCD, which outperformed sceHCD in the accuracy of identification. Herein, we integrated the combinatorial peptide ligand library (CPLL) into EThcD-sceHCD and compared the performance of different mass spectrometry dissociation methods (EThcD-sceHCD, EThcD, and sceHCD) in the intact N-glycopeptide analysis of prostate cancer plasma. The results illustrated that EThcD-sceHCD was better than EThcD and sceHCD in the number of identified intact N-glycopeptides (two-folds). A combination of sceHCD and EThcD-sceHCD methods can cover almost all glycoproteins (96.4%) and intact N-glycopeptides (93.6%), indicating good complementarity between the two. Our study has great potential for medium- and low-abundance plasma glycoprotein biomarker discovery.

MO711: Evaluation of an in Vitro Co-Culture Model for Studying Modulation of Cross-Talk between Endothelial and Mesothelial Cells by Cytoprotective Additives in Peritoneal Dialysis Fluids

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a life-saving renal replacement therapy. The composition of all currently available peritoneal dialysis fluids (PDF) triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization and diabetes-like damage of vessels, eventually leading to failure of the technique. In vitro and in vivo studies have shown that cytoprotective additives [e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)] to PDF reduce peritoneal damage in mesothelial (MC) and endothelial (EC) cells. However, it is yet unclear if potential cross talk between cells present in the peritoneal membrane mediates the observed modulation of PD-associated changes. Currently, there is no model system available to study relevant processes in these cells when in close proximity. Here, we aimed to develop a co-culture model for investigating cell-to-cell communication in a PD-relevant setting using cellular proteome and secretome analyses and investigate the effects of cytoprotective additives. METHOD For modelling the peritoneal membrane in vitro, MCs and ECs were co-cultured in transwell plates. MCs were grown in the upper compartment and primary microvascular ECs were grown in the lower compartment. PDF with or without additives, was added to the upper compartment to expose MCs directly, while the ECs below were kept in medium. Cellular proteome and secretome profiles were analysed for both cell types cultured individually or together by a quantitative mass spectrometry approach. Prior to analysis of the secretome, a combinatorial peptide ligand library coupled to beads was employed to enrich low abundant proteins and deplete high abundant proteins. RESULTS Proteome analysis revealed perturbation of major cellular processes including cytoskeleton reorganization, stress response, and regulation of cell death that characterize PDF cytotoxicity. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to individual cultures. Several pathways relevant for PD, such as VEGF signaling in ECs or oxidative stress response in MCs were found only under co-culture conditions. Combined analysis of proteome and secretome showed specific ligand–receptor pairs expressed only under co-culture conditions regulating some of these pathways. Differentially regulated cellular and secreted proteins after PDF exposure were related to processes like angiogenesis, immune response, or and extracellular processes such as integrin signaling. Addition of 10 mM LiCl to PDF led to modulation of VEGF and oxidative stress response pathways in ECs as well as ligand–receptor pairs involved in these pathways, possibly explaining MC–EC crosstalk during PDF exposure of these cells and the cytoprotective effect of LiCl. CONCLUSION Harmful effects of PDF on MCs may also affect ECs, as demonstrated by our data. Interestingly, both cells type react differently when co-cultured compared to individual culture models, showing the importance of models that allow cell type interaction to mimic the in vivo conditions. We identified potential signaling axes between the cell types that could explain pathophysiological changes of the peritoneal membrane during PD treatment. We have also elucidated potential mechanisms by which the cytoprotective additive LiCl may modulate crosstalk between MCs and ECs to counteract adverse PDF effects in the local peritoneal environment. Linking local peritoneal damage with systemic vasculopathy through perturbations of PD-induced cellular crosstalk, may identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.

Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

Combinatorial peptides: A library that continuously probes low-abundance proteins.

After a decade of experimental applications, it is the objective of this review to make a point on combinatorial peptide ligand libraries dedicated to low-abundance proteins from animals to plants and to microorganism proteomics. It is, thus, at the light of the recent technical developments and applications that we will examine the state of the art, its usage within the scientific community, and its openness to unexplored fields. The improvements of the methodology and its implementation in connection with analytical determinations of combinatorial peptide ligand library (CPLL)-treated samples are extensively reviewed and commented upon. Relevant examples covering few critical aspects describe the performance of the technology. Finally, a reflection on the technological future is attempted in particular by involving new concepts adapted to the limited availability of certain biological samples.

MO684A CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PD FLUIDS ON THE SECRETOME OF MESOTHELIAL AND ENDOTHELIAL CELLS

Abstract Background and Aims The composition of all currently available peritoneal dialysis fluids (PDF) triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels of the peritoneal membrane, eventually leading to technique failure. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of such additives on secretome-mediated signalling between cell-types of the peritoneal membrane which are relevant in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial (MC) and endothelial cells (EC) were co-cultured in transwell plates. MC were grown in the upper compartment and primary microvascular cells were grown in the lower compartment. MC were exposed to PDF with or without cytoprotective compounds (8 mM AlaGln in glucose-based PDF 3.86% or 10 mM LiCl in icodextrin-based PDF), while EC below were kept in medium. Cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release, neutral red uptake and cell morphology. Proteome and secretome profiles were analysed for both cell-types in co-culture or separately with an isobaric-tag labelling approach with a multiplexed liquid chromatography/mass spectrometry (LC-MS) approach. Prior to analysis of the secretome a bead-based equalizer approach based on a combinatorial peptide ligand library (CPLL) was performed to enrich low abundant proteins. Results EC injury after PD-fluid exposure of MC was decreased with the addition of AlaGln or LiCl, showing a link between the individual cell outcomes. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton re-organization, which characterize PDF cytotoxicity. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. We were able to identify and quantify 334 secreted proteins in the co-culture system. The secretome analysis showed variation in several clinically relevant proteins and important extracellular processes such as extracellular matrix reorganization, vesicle transport or collagen deposition. Comparison to previously published abundance profiles of omental arteriolar proteins from paediatric PD patient and age-matched controls confirmed overlapping protein regulation between endothelial cells in vitro and in vivo. Conclusion This study shows that harmful effects of PDF-stressed MC also affect EC and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the close proximity of different cell types in the peritoneal membrane for testing direct and indirect effects of cytoprotective additives. Characterisation of PD-induced perturbations may allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS.

BackgroundSnake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed.MethodsShotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL).ResultsThis avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5’-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2).ConclusionThese findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.

ITRAQ-based proteomic analysis reveals potential serum biomarkers of allergic and nonallergic asthma.

Asthma is heterogeneous disease with different phenotypes, endotypes and severities. Definition of these subgroups requires the identification of biomarkers in biological samples, and serum proteomics is a useful and minimally invasive method for this purpose. Therefore, the aim of this study was to detect serum proteins whose abundance is distinctively associated with different asthma phenotypes (allergic vs nonallergic) or severities. For each group of donors (32 healthy controls, 43 allergic rhinitis patients and 192 asthmatics with different phenotypes and severities), we generated two pools of sera that were analysed by a shotgun MS approach based on combinatorial peptide ligand libraries and iTRAQ-LC-MS/MS. MS analyses identified 18 proteins with a differential abundance. Functional/network study of these proteins identified key processes for asthma pathogenesis, such as complement activation, extracellular matrix organization, platelet activation and degranulation, or post-translational protein phosphorylation. Furthermore, our results highlighted an enrichment of the "Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)" route in allergic asthma and the lectin pathway of complement activation in nonallergic asthma. Thus, several proteins (eg IGFALS, HSPG2, FCN2 or MASP1) displayed a differential abundance between the different groups of donors. Particularly, our results revealed IGFALS as a useful biomarker for moderate-severe allergic asthma. Our data suggest a set of serum biomarkers, especially IGFALS, capable of differentiating allergic from nonallergic asthma. These proteins reveal different pathophysiological mechanisms and may be useful in the future for diagnosis, prognosis or targeted therapy purposes.

Insight of low-abundance proteins in rice leaves under Cd stress using combinatorial peptide ligand library technology.

Low-abundance proteins (LAPs) play a very important role in interaction, regulation, and metabolism of plant biological processes. A combinatorial peptide ligand library (CPLL) can solve the problem of high-abundance proteins (HAPs) masking LAPs and enlarging the dynamic range of protein concentrations perfectly and be considered as one of the most advanced approaches for plant proteomics research. In this paper, a proper CPLL method to rice leaf proteins was established for the first time and 1056 proteins were identified in rice leaf extracts, and 624 (59.1%) LAPs were newly detected after CPLL. Based on this technology, we detected the response of rice to Cd stress and analyzed the differential LAPs and the biological significance of misexpressed proteins before and after Cd stress by bioinformatics analysis. An important contribution has also been made to a better understanding of the complex mechanisms by which rice adapts to Cd stress. Graphical abstract.

Comparison of accompanying proteins in different therapeutic human serum albumin preparations

Pharmaceutical human serum albumin products are manufactured from donated human plasma and may contain up to 5% accompanying non-albumin proteins. It has been reported that albumin preparations manufactured by different pharmaceutical companies differed in the degree of posttranslational modifications, the redox state as well as antioxidant properties of albumin, whereas the composition of the accompanying proteins has never been comparatively analyzed. In this study, a non-targeted mass spectrometric approach was used for label-free quantification and comparison of different pharmaceutical albumin preparations. Haptoglobin and a few other proteins accounted for approximately 80% of the accompanying proteins in all products tested. Low abundance proteins were enriched by means of a combinatorial peptide ligand library (ProteoMiner, Bio-Rad). Significant differences between the amounts of several mainly low abundance proteins, such as complement factors, were observed indicating differences in the manufacturing processes of the pharmaceutical companies. The removal of the stabilizers octanoate and N-acetyltryptophan from albumin solutions using the charcoal-based Hepalbin adsorbent simultaneously reduced the accompanying proteins. For therapy evaluation of albumin preparations, the variable composition of the accompanying proteins in different albumin products should be taken into account in addition to the known heterogeneity of the albumin protein itself.

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss).

The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.

Detection of Plant Low-Abundance Proteins by Means of Combinatorial Peptide Ligand Library Methods.

The detection and identification of low-abundance proteins from plant tissues is still a major challenge. Among the reasons are the low protein content, the presence of few very high-abundance proteins, and the presence of massive amounts of other biochemical compounds. In the last decade numerous technologies have been devised to resolve the situation, in particular with methods based on solid-phase combinatorial peptide ligand libraries. This methodology, allowing for an enhancement of low-abundance proteins, has been extensively applied with the advantage of deciphering the proteome composition of various plant organs. This general methodology is here described extensively along with a number of possible variations. Specific guidelines are suggested to cover peculiar situations or to comply with other associated analytical methods.

Glyco-CPLL: An Integrated Method for In-Depth and Comprehensive N-Glycoproteome Profiling of Human Plasma.

N-glycoproteins are involved in various biological processes. Certain distinctive glycoforms on specific glycoproteins enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential for a new biomarker discovery. The absence of suitable analytical methods for in-depth and large-scale analyses of low-abundance plasma glycoproteins makes it challenging to investigate the role of glycosylation. In this study, we developed an integrated method termed Glyco-CPLL, which integrates combinatorial peptide ligand libraries, high-pH reversed-phase prefractionation, hydrophilic interaction chromatography, trypsin and PNGase F digestion, shotgun proteomics, and various analysis software (MaxQuant and pGlyco2.0) for the low-abundance plasma glycoproteomic profiling. Then, we utilized the method to perform a comparative study and to explore papillary thyroid carcinoma-related proteins and glycosylations with reference to healthy controls. Finally, a large and comprehensive human plasma N-glycoproteomic database was established, containing 786 proteins, 369 N-glycoproteins, 862 glycosites, 171 glycan compositions, and 1644 unique intact N-glycopeptides. Additionally, several low-abundance plasma glycoproteins were identified, including SVEP1 (∼0.54 ng/mL), F8 (∼0.83 ng/mL), and ADAMTS13 (∼1.2 ng/mL). These results suggest that this method will be useful for analyzing plasma intact glycopeptides in future studies. Besides, the Glyco-CPLL method has a great potential to be translated to clinical applications. Data are available via ProteomeXchange with identifier PXD016428.

Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method and to compare it with two commercial kits, including HSA and IgG immunodepletion (IMDP) and combinatorial peptide ligand libraries (CPLL), using untreated plasma as a control condition. By varying the temperature and the incubation period, the optimal condition was found as treatment at 95°C for 20 min, which maintained about 1% recovery yield of soluble proteins. Consistency and reproducibility of thermal treatment-derived plasma subproteome were checked by two-dimensional electrophoresis. The coefficient of variation regarding protein spot numbers was less than 10% among three independent specimens. Highly abundant protein depletion of the thermal treatment was evaluated by immunoblotting against HSA and IgG as compared to the untreated plasma, IMDP, and CPLL. Multidimensional comparison based on 489 unique peptides derived from the label-free quantitative mass spectrometry revealed that the thermal treatment, IMDP, and CPLL provided distinct sets of plasma subproteome compared to untreated plasma, and these appeared to be complementary to each other. Comparing the characteristics of the three procedures suggested that thermal treatment was more cost-effective and less time-consuming than IMDP and CPLL. This study proposes the use of thermal treatment as a reliable and cost-effective method for plasma prefractionation which provides benefits to large-scale proteomic projects and biomarker studies.

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage

Cerebrospinal fluid (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 complicated 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 affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC–MS/MS analysis. LC–MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification 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 flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (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.

Progress in farm animal proteomics: The contribution of combinatorial peptide ligand libraries

The present review covers proteomics discoveries in farm animals using combinatorial peptide ligand libraries (CPLLs). These libraries enhance the identification of low-abundance proteins by compressing the dynamic range of the protein concentration. This technology can be applied in multiple biological fluids, such as plasma or serum, follicular and cerebrospinal fluids, urine, saliva, tears seminal fluid, milk whey and even lymph. The discovery of low-abundance proteins contributes to the continuous monitoring for animal pathologies, which allows early treatment and disease prevention. Additionally, evidencing rare proteins in animal products used for human consumption can help in assessing their nutritional properties and their positive or negative effects on human health. SignificanceMany investigations are made on the identification of proteins of farm animals. Protein concentration changes under abnormal stressful conditions as a sign of a first physiological reaction. Many of these protein changes are of low-abundance and their detection is dependent on enrichment technologies. These changes are potentially correlated with the type of stressor and may determine actions to preserve the animal welfare.

Combinatorial Peptide Ligand Library-Based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins.

Phosphotyrosine (pY) serves as a docking site for the recognition proteins containing pY-binding (pYB) modules, such as the SH2 domain, to mediate cell signal transduction. Thus, it is vital to profile these binding proteins for understanding of signal regulation. However, identification of pYB proteins remains a significant challenge due to their low abundance and typically weak and transient interactions with pY sites. Herein, we designed and prepared a pY-peptide photoaffinity probe for the robust and specific enrichment and identification of its binding proteins. Using SHC1-pY317 as a paradigm, we showed that the developed probe enables to capture target protein with high selectivity and remarkable specificity even in a complex context. Notably, we expanded the strategy to a combinatorial pY-peptide-based photoaffinity probe by using combinatorial peptide ligand library (CPLL) technique and identified 24 SH2 domain proteins, which presents a deeper profiling of pYB proteins than previous reports using affinity probes. Moreover, the method can be used to mine putative pYB proteins and confirmed PKN2 as a selective binder to pY, expanding the repertoire of known domain proteins. Our approach provides a general strategy for rapid and robust interrogating pYB proteins and will promote the understanding of the signal transduction mechanism.

Proteomic fingerprinting of apple fruit, juice, and cider via combinatorial peptide ligand libraries and MS analysis.

Combinatorial peptide ligand libraries coupled to MS was applied to extensively map the proteome of apple fruit, and to detect its presence in commercial apple juice and cider to evaluate their authenticity and genuineness. Using the Uniprot_Malus database, 96 proteins were detected in apples, among which 30 proteins were specifically captured via combinatorial peptide ligand libraries. Next, three proteins, previously recognized in fruits, were found in apple juice, which were involved in cellular metabolism of fruit maturation and in allergenic reactions. On the other hand, only one Malus allergen was identified in cider beads eluate, demonstrating that the industrial processes did not prevent any negative effects in sensitive subjects. Thus, the present study not only increases the knowledge of the apple proteome but also offers a reliable analytical method to assess quality and genuineness of commercial products, which could be also used to inform consumers about the presence of allergens.

All Cats are Gray in the Dark: Enrichment/Depletion Approaches for Biomarker Discovery on Felis catus Plasma

In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.