Nano-ultra Performance Liquid Chromatography Research Articles

Proteomic approach towards identification of seminal fluid biomarkers from individuals with severe oligozoospermia, cryptozoospermia and non-obstructive azoospermia: a pilot study.

Infertility becomes a global problem that affects to the same extent females and males. As reasons of male infertility can differ among individuals, the accurate diagnostics is essential for effective treatment. The most problematic both in diagnostics and in treatment are disturbances of spermatogenesis. Seminal fluid is rich in proteins that potentially can serve as markers for male infertility and among them, markers of spermatogenesis which are highly desired. To find biomarkers of spermatogenesis, we applied comparative proteomics using nano ultra performance liquid chromatography and tandem mass spectrometry (nanoUPLC-MS/MS) followed by single-sample Western blotting (WB) using seminal fluid samples from males with different types of infertility including non-obstructive azoospermia (NOA), cryptozoospermia (C) and severe oligozoospermia (SO). Then, the extensive survey on the identified proteins and their function in male reproductive system has been done. The proteomic approach has enabled to identified five seminal fluid proteins being potential markers of spermatogenesis disorders: ADGRG2, RAB3B, LTF, SLC2A3 and spermine synthase (SMS). Among them ADGRG2 seems to be strongly involved in male infertility. In addition, WB indicated that the distribution of LTF, SLC2A3 and SMS was not coherent among the individuals, especially in a group with NOA. Functional annotation analysis and search in proteomics databases revealed that vast majority of the proteins originated from extracellular environment. The presented data point out several proteins that potentially can become biomarkers of male infertility. The data suggest, however, different mechanisms behind the male infertility indicating that the etiology is more complex. We assume that recognition of these mechanisms may lead to the creation of specific protein panel helpful in the management of male infertility and therefore, further studies are required.

Proteomic profiling of Klebsiella pneumoniae carbapenemase (KPC)-producer Klebsiella pneumoniae after induced polymyxin resistance.

Aim: To elucidate the changes in protein expression associated with polymyxin resistance in Klebsiella pneumoniae, we profiled a comparative proteomic analysis of polymyxin B-resistant mutants KPC-2-producing K. pneumoniae, and of its susceptible counterparts. Material & methods: Two-dimensional reversed phase nano ultra-performance liquid chromatography mass spectrometry was used for proteomic analysis. Results: Our results showed that the proteomic profile involved several biological processes, and we highlight the downregulation of outer membrane protein A (OmpA) and the upregulation of SlyB outer membrane lipoprotein (conserved protein member of the PhoPQ regulon) and AcrA multidrug efflux pump in polymyxin B-resistant strains. Conclusion: Our results highlight the possible participation of the SlyB, AcrA and OmpA proteins in the determination of polymyxin B heteroresistance in KPC-2-producing K. pneumoniae.

Impact of the Trophic Effects of the Secretome From a Multistrain Probiotic Preparation on the Intestinal Epithelia

Probiotics are defined as live, nonpathogenic bacteria that confer health benefits beyond their nutritional value. In particular, VSL#3 exhibits demonstrated efficacy in the management of diseases characterized by an increased intestinal permeability. Our study aimed to understand how VSL#3 promotes gut health by secreting bioactive factors and identify which human pathways are modulated by secretome derived from the VSL#3 formula. Two different lots of VSL#3 were used, and Caco-2 cell line was treated with conditioned media (CM) prepared using 1 g of the probiotic formula. We evaluated the effects of the probiotics on cellular proliferation and apoptosis by cytometry and the expression of tight junction proteins by western blotting. A proteomics analysis of both culture media and the whole proteome of Caco-2 cells treated with VSL#3-CM was performed by nano-ultra performance liquid chromatography - tandem mass (nUPLC MS/MS) spectrometry. The probiotic formula increased cell proliferation, decreased cellular apoptosis cells, and increased re-epithelialization in the scratch assay. Several peptides specifically synthetized by all the species within the probiotic preparation were recognized in the proteomics analysis. Human proteins synthesized by CaCo-2 cells were also identified. To our knowledge, this manuscript describes the first evaluation of the probiotic secretome, and the results showed that the improvement in intestinal barrier functions induced by probiotics seems to be accompanied by the modulation of some human cellular pathways.

Preliminary Study on the Biological Markers for I-IIb Stage Non-small Cell Lung Cancer Based on a Serum-peptidomics

背景与目的非小细胞肺癌是发病率最高的一种肺恶性肿瘤，早发现、早诊断、早治疗是其治疗原则，因其发病隐匿，缺乏早期筛查的特异性标志物，多数患者就诊时已处于晚期阶段，导致其5年生存率较低，预后不理想。探索一种敏感的生物学标志物，是目前肺癌诊疗的重点。本文基于差异多肽组学技术，旨在筛选Ⅰ期-Ⅱb期非小细胞肺癌的血清生物标志物。方法利用纳升超高效液相色谱联合四级杆轨道阱质谱技术，比较和筛选正常健康人群、肺部良性病变患者、非小细胞肺癌患者血清中的差异多肽片段，通过对目标肽段的鉴别和定量分析，探寻非小细胞肺癌早期诊断的肿瘤生物标志物。结果通过与人类蛋白质组数据库进行比对，共鉴定到545个多肽，分别来自118个蛋白。将各组样本中肽段的谱图数进行比较，共筛选出201个差异多肽，经定量分析发现峰面积的变异系数（coefficient of variation, CV）≤30%的目标肽段共计7个，其中在各组间呈趋势变化的有2条肽段，分别是肺癌组中表达下调的肽段QGAKIPKPEASFSPR和肺癌组中表达上调的肽段CDDYRLC，它们分别来自于中间α球蛋白抑制因子H4蛋白（ITIH4）和基质γ-羧基谷氨酸蛋白（MGP）。结论将血清多肽组学用于筛选肿瘤标志物的研究，可为非小细胞肺癌的早期诊断提供新线索，其中ITIH4与MGP蛋白水解的特异性肽段是潜在早期非小细胞肺癌患者的血清标志物。

Immunoglobulin G N-Glycans as Potential Postgenomic Biomarkers for Hypertension in the Kazakh Population.

Next-generation (postgenomic) biomarkers from the nascent field of glycomics now offer fresh vistas for innovation in chronic disease biomarkers and system diagnostics in clinical medicine. Our previous work has shown an association between hypertension and immunoglobulin G (IgG) glycome composition, suggesting that individual variation in N-glycosylation of IgG might contribute to hypertension pathogenesis. The present study examined, for the first time to the best of our knowledge, the IgG N-glycans as potential biomarkers for hypertension in the Kazakh population. The profile of 60 N-glycopeptides of IgG subclass isolated from plasma samples of 150 Kazakh study participants was analyzed by nano ultra-performance liquid chromatography with mass spectrometry. Fourteen IgG subclass-specific Fc N-glycopeptide structures, along with one derived glycosylation trait in subclasses IgG2/3 and IgG4, were found to correlate with systolic blood pressure and/or diastolic blood pressure. For differentiation of hypertension and healthy status in the Kazakh population sample, the receiver operating characteristic curve analysis showed that the performance of the model, including nine IgG N-glycans, was greater than the traditional gender, age, and body mass index based model (p < 0.05). This study indicates that alteration in Fc N-glycopeptide profiles of plasma IgG subclasses is associated with blood pressure status in the Kazakh population. IgG N-glycosylation profiles may serve as potential biomarkers for hypertension in the Kazakhs, thus contributing to move toward personalized medicine. Further studies of postgenomic glycomic biomarkers in cardiovascular and chronic diseases are timely and called for.

A Quantitative Assay of In Vivo Cleavage of Von Willebrand Factor By ADAMTS13 Reveals Excessive Cleavage in Type 2B Von Willebrand Disease and Diminished Cleavage in Thrombotic Thrombocytopenic Purpura

Von Willebrand Factor (VWF) is a large glycoprotein that plays a crucial role in hemostasis by enabling platelets to adhere to sites of vascular injury, particularly under conditions of high shear stress. The multimeric structure of VWF is central to its activity; VWF is most active in its ultra-large multimer form (ULVWF). The size of plasma VWF is regulated by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Tyr1605-Met1606 peptide bond within the A2 domain. Abnormal VWF cleavage has been implicated in a wide range of thrombotic and bleeding disorders, including thrombotic thrombocytopenic purpura (TTP) and Type 2 von Willebrand disease (VWD). These two diseases represent extreme phenotypes of VWF proteolysis-the former with too little cleavage and the latter with too much cleavage. ADAMTS13 proteolysis of VWF is currently assessed by either multimer gel analysis or ADAMTS13 activity assays. High-resolution agarose gels give an estimate of the extent to which a particular band represents cleaved vs native VWF. This method, however, is not quantitative and cannot distinguish whether a particular cleavage product is produced by ADAMTS13 or by another protease. Alternatively, ADAMTS13 activity assays are indirect measures of VWF cleavage, used especially to diagnose TTP. These assays measure the extent to which a subject's ADAMTS13 can cleave an exogenous substrate, usually a small peptide, and do not determine the extent of endogenous VWF cleavage. Therefore, we developed a mass spectrometry-based method to directly quantify cleavage of the Tyr1605-Met1606 peptide bond in plasma VWF. Here, we report quantification of VWF cleavage in plasma from normal donors, and TTP and VWD patients.Methods: VWF was isolated from plasma by immunoprecipitation using polyclonal VWF antibody and then digested with trypsin. Tryptic peptides, including the ADAMTS13-cleaved peptide and a control peptide, were quantified by nano ultra-performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using heavy isotope-labelled peptides as internal standards. The extent of VWF cleavage by ADAMTS13 was determined as the concentration ratio of ADAMTS13-cleaved peptide to a control peptide representative of total VWF, and expressed as percent cleavage.Results: Given that the plasma concentration of VWF is relatively low, we first optimized conditions for sample preparation and analysis. Using NanoLC-MS/MS with isotopic dilution, we quantified ADAMTS13 cleavage of VWF in plasma samples (10-100 µl) containing 100-500 ng of VWF. We tested the reproducibility of the measurement using pooled normal plasma. The quantification was highly reproducible, with a wide linear range. VWF cleavage in pooled normal plasma was 4.9% ± 0.3% (mean ± SD, n=6), indicating that one VWF monomer in 20 is cleaved, on average. Cleavage was much higher in plasma from type 2B VWD patients (n=5), ranging from 15-30% depending on mutations. In contrast, we observed only 2.1-3.2% (n=6) cleavage in plasma VWF from TTP patients.Summary: This is the first report of quantification of ADAMTS13-mediated VWF cleavage in plasma. In normal plasma, approximately 5% of VWF A2 domains are cleaved, whereas cleavage was reduced to approximately 60% of normal on average in 6 TTP patients, and increased up to 6 times normal in patients with Type 2B VWD. In TTP, this level of proteolysis would be expected to result in accumulation of ULVWF multimers, as only between 1 in 50 to 1 in 30 monomers were cleaved. In type 2B VWD, by contrast, proteolysis may play a much larger role in the lack of larger multimers than previously appreciated, as up to 1 in 3 monomers were cleaved. This method should also prove useful in conditions such as severe malaria, in which it has been suggested that VWF cleavage is impaired, but this is not necessarily accompanied by low ADAMTS13 activity as measured with a small peptide substrate. DisclosuresNo relevant conflicts of interest to declare.

Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study.

Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a systematic investigation of protein repertoire in controlled platelet preparation. Platelets have been extracted from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. The quality of the preparation was assessed by observation and enumeration in a Bürker chamber with a conventional tissue culture microscope. To characterize human platelets proteome we analysed the pool of purified platelets combining two proteomic approaches: 2-DE separation combined with Mass Spectrometry and nanoscale ultra performances LC-MS(E) shotgun proteomics experiments. The 2D gel analysis leads an average of 1900 protein spots, after the filtering of "noise" and "false positive" spots, over 500 were selected to be eligible for further analysis given their optimal spot quality value. To perform the analysis by ion accounting shotgun proteomic approach, based on nano ultra performance liquid chromatography (nUPLC) coupled to MS(E) processing of continuum LC-MS data, the same pool of samples was subject to liquid phase tryptic digestion and the peptide obtained used for the experiments. All the data obtained were analysed using ProteinLynx GlobalServer v2.3 (PLGS, Waters). Three analytical replicates run were acquire in high/low energy modes and associated to a human protein database returning the identification of 100 distinct genes. Comparative analysis of the Gene Ontology has been performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches. The overall molecular function classification revealed differences between the two proteomic approaches. In particular, we found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun).

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC-HRMS/MS.

Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable relevance in sports drug testing. Such peptides have been used to manipulate several potential performance-enhancing processes in the athlete's body and include for example growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin), synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano growth factors (human MGF, modified human MGF, 'full-length' MGF). A combined initial test method using one analytical procedure is a desirable tool in doping controls and related disciplines as requests for higher sample throughput with utmost comprehensiveness preferably at reduced costs are constantly issued. An approach modified from an earlier assay proved fit-for-purpose employing pre-concentration of all target analytes by means of ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high performance liquid chromatography (UHPLC) separation, and subsequent detection by means of high resolution tandem mass spectrometry. The method was shown to be applicable to blood and urine samples, which represent the most common doping control specimens. The method was validated considering the parameters specificity, recovery (11-69%), linearity, imprecision (<25%), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma), and ion suppression. The analysis of administration study samples for insulin degludec, detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the method.

Lysine acetylproteome analysis suggests its roles in primary and secondary metabolism in Saccharopolyspora erythraea

Lysine acetylation is a dynamic, reversible posttranslational modification that is known to play an important role in regulating the activity of many key enzymes in bacteria. Acetylproteome studies have been performed on some bacteria. However, until now, there have been no data on Actinomycetes, which are the major producers of therapeutic antibiotics. In this study, we investigated the first acetylproteome of the erythromycin-producing actinomycete Saccharopolyspora erythraea using a high-resolution mass spectrometry-based proteomics approach. Using immune-affinity isolation of acetyl-peptides with an anti-acetyllysine antibody followed by nano ultra performance liquid chromatography tandem mass spectroscopy (nanoUPLC-MS/MS) analysis, we identified 664 unique lysine-acetylated sites on 363 proteins. Acetylated proteins are involved in many biological processes such as protein synthesis, glycolysis/gluconeogenesis, citric acid (TCA) cycle, fatty acid metabolism, secondary metabolism, and the feeder metabolic pathways of erythromycin synthesis. We characterized the acetylproteome and analyzed in detail the impact of acetylation on diverse cellular functions according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Four motif sequences surrounding the acetylation sites (K(AC)H, K(AC)Y, K(AC)XXXXR, and K(AC)XXXXK) were found in the S. erythraea acetylproteome. These findings suggest that abundant lysine acetylation occurs in Actinomycetes, expand our current knowledge of the bacterial acetylproteome, and provide insight into the regulatory function of acetylation in primary and secondary metabolism.

Photocatalytic degradation of rosuvastatin: Analytical studies and toxicity evaluations

Photocatalytic degradation of rosuvastatin, which is a drug that has been used to reduce blood cholesterol levels, was studied in this work employing ZnO as catalyst. The experiments were carried out in a temperature-controlled batch reactor that was irradiated with UV light. Preliminary the effects of the photocatalyst loading, the initial pH and the initial rosuvastatin concentration were evaluated. The experimental results showed that rosuvastatin degradation is primarily a photocatalytic process, with pseudo-first order kinetics. The byproducts that were generated during the oxidative process were identified using nano-ultra performance liquid chromatography tandem mass spectrometry (nano-UPLC–MS/MS) and acute toxicity tests using Daphnia magna were done to evaluate the toxicity of the untreated rosuvastatin solution and the reactor effluent.

A Nano Ultra-Performance Liquid Chromatography–High Resolution Mass Spectrometry Approach for Global Metabolomic Profiling and Case Study on Drug-Resistant Multiple Myeloma

Global metabolomics relies on highly reproducible and sensitive detection of a wide range of metabolites in biological samples. Here we report the optimization of metabolome analysis by nanoflow ultraperformance liquid chromatography coupled to high-resolution orbitrap mass spectrometry. Reliable peak features were extracted from the LC-MS runs based on mandatory detection in duplicates and additional noise filtering according to blank injections. The run-to-run variation in peak area showed a median of 14%, and the false discovery rate during a mock comparison was evaluated. To maximize the number of peak features identified, we systematically characterized the effect of sample loading amount, gradient length, and MS resolution. The number of features initially rose and later reached a plateau as a function of sample amount, fitting a hyperbolic curve. Longer gradients improved unique feature detection in part by time-resolving isobaric species. Increasing the MS resolution up to 120000 also aided in the differentiation of near isobaric metabolites, but higher MS resolution reduced the data acquisition rate and conferred no benefits, as predicted from a theoretical simulation of possible metabolites. Moreover, a biphasic LC gradient allowed even distribution of peak features across the elution, yielding markedly more peak features than the linear gradient. Using this robust nUPLC-HRMS platform, we were able to consistently analyze ~6500 metabolite features in a single 60 min gradient from 2 mg of yeast, equivalent to ~50 million cells. We applied this optimized method in a case study of drug (bortezomib) resistant and drug-sensitive multiple myeloma cells. Overall, 18% of metabolite features were matched to KEGG identifiers, enabling pathway enrichment analysis. Principal component analysis and heat map data correctly clustered isogenic phenotypes, highlighting the potential for hundreds of small molecule biomarkers of cancer drug resistance.

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography–tandem mass spectrometry coupled with microscale labeling

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

Proteomic Profiling for Peritoneal Dialysate: Differential Protein Expression in Diabetes Mellitus

Peritoneal dialysis (PD) is an increasingly accepted modality of renal replacement therapy. It provides the advantages of having a flexible lifestyle, stable hemodynamics, and better preservation of residual renal function. To enhance our understanding of the peritoneal dialysate of diabetes mellitus (DM), peritoneal dialysate proteins were identified by two-dimensional gel electrophoresis (2DE) combined with reverse-phase nano-ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-nano-UPLC-ESI-MS/MS) followed by peptide fragmentation patterning. To validate the differential proteins, ELISA and Western blotting analyses were applied to detect candidate proteins that may be related to DM. We performed 2DE on the peritoneal dialysate samples, with detection of more than 300 spots. From this, 13 spots were excised, in-gel digested, and identified by RP-nano-UPLC-ESI-MS/MS. Ten of these showed significant differential expression between the DM and chronic glomerulonephritis (CGN) peritoneal dialysate samples. In this study, we conducted a comparative proteomic study on these two groups of dialysate that may provide evidence for understanding the different peritoneal protein changes. These proteins may not be new biomarkers; however, they may indicate a situation for possible drug treatment and can be the predictors of peritonitis for a validation study in the future.

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MSE). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus non-infected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery.

Label-free Quantitative Proteomics Reveals Differentially Regulated Proteins Influencing Urolithiasis

Urinary proteins have been implicated as inhibitors of kidney stone formation (urolithiasis). As a proximal fluid, prefiltered by the kidneys, urine is an attractive biofluid for proteomic analysis in urologic conditions. However, it is necessary to correct for variations in urinary concentration. In our study, individual urine samples were normalized for this variation by using a total protein to creatinine ratio. Pooled urine samples were compared in two independent experiments. Differences between the urinary proteome of stone formers and nonstone-forming controls were characterized and quantified using label-free nano-ultraperformance liquid chromatography high/low collision energy switching analysis. There were 1063 proteins identified, of which 367 were unique to the stone former groups, 408 proteins were unique to the control pools, and 288 proteins were identified for comparative quantification. Proteins found to be unique in stone-formers were involved in carbohydrate metabolism pathways and associated with disease states. Thirty-four proteins demonstrated a consistent >twofold change between stone formers and controls. For ceruloplasmin, one of the proteins was shown to be more than twofold up-regulated in the stone-former pools, this observation was validated in individuals by enzyme-linked immunosorbent assay. Moreover, in vitro crystallization assays demonstrated ceruloplasmin had a dose-dependent increase on calcium oxalate crystal formation. Taken together, these results may suggest a functional role for ceruloplasmin in urolithiasis.

Analysis of autophosphorylation sites in the recombinant catalytic subunit alpha of cAMP-dependent kinase by nano-UPLC–ESI–MS/MS

The catalytic subunit of recombinant wild-type cyclic adenosine monophosphate-dependent protein kinase A (PKA) has been analyzed by a combination of 1D gel electrophoresis, in-gel digestion by trypsin, chymotrypsin, or endoproteinase AspN, and nano-ultraperformance liquid chromatography--MS/MS. The MS/MS spectra were annotated by MASCOT and the annotations were manually controlled. Using Ga(III)-immobilized metal ion affinity chromatography (IMAC), in addition to the four established autophosphorylation sites of the catalytic subunit of recombinant PKA, pSer10, pSer139, pThr197, and pSer338, six new phosphorylated residues have been characterized--pSer14, pThr48, pSer53, pSer212, pSer259, and pSer325. The established phosphorylation sites all are part of a PKA consensus motif and were found to be almost completely modified. In contrast, the newly detected sites were only partially phosphorylated. For estimation of their degree of phosphorylation, a method based on signal intensity measurements was used. For this purpose, signal intensities of all phospho- and non-phosphopeptides containing a particular site were added for estimation of site-specific phosphorylation degrees. This addition was performed over all peptides observed in the different digestion experiments, including their different charge states. pThr48 and pSer259 are located within PKA consensus motifs and were observed to be phosphorylated at 20% and 24%, respectively. pSer14 and pSer53 are located within inverted PKA consensus motifs and were found to be phosphorylated around 10% and 15%, respectively. The sequence environments of pSer212 and pSer325 have no similarity to the PKA consensus motif at all and were observed to be phosphorylated at about 5% or lower. All newly observed phosphorylation sites are located at the surface of the native protein structure of the PKA catalytic subunit. The results add new information on the theme of site-specific (auto)phosphorylation by PKA.

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography–tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.

Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the ubiquitin proteasome pathway

Objectives:To determine if peripheral blood monocytes from patients with ankylosing spondylitis (AS) differed in protein expression compared to rheumatoid arthritis (RA) and healthy controls (HC).Methods:Monocyte protein expression was characterised by...