In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

Partial hepatectomy induced liver proteome changes in mice

Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases. However, at present little is known about therapeutic interventions to enhance this regenerative process. Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment. Here we propose a broad-range proteomic approach to this problem that makes possible the simultaneous study of different signaling and metabolic pathways on the liver proteome. Changes in protein expression in mouse livers (n = 5 per group) at 6 h and 12 h after partial hepatectomy and sham operation, as compared to untreated controls, were analyzed using two-dimensional gel electrophoresis, mass spectrometry (MS), and mass fingerprinting. Twelve proteins, identified by MS, were up-regulated by at least 2-fold after partial hepatectomy. These included adipose differentiation-related protein, gamma-actin, enoyl coenzyme A hydratase 1, serum amyloid A and eukaryotic translation initiation factor 3. These results indicate that liver regeneration following partial hepatectomy affects various signaling and metabolic pathways.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery

Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.     

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

Mass Spectrometry and Proteomics 2018: the Mass Spectrometry Society of Japan,Japanese Proteomics Society,and Asia-Oceania Human Proteome Organization

ABSTRACT

Introduction: The mass spectrometry society of Japan, Japanese proteomics society, and Asia–Oceania human proteome organization held the conference ‘Mass Spectrometry and Proteomics 2018’ in Osaka, Japan, on May 15–18, 2018. This international conference focused on cutting edge technologies and their applications in a variety of research fields such as agriculture, material science, environmental factors, and clinical applications. An overview of the conference and a summary of the major lectures are reported here.

Expert commentary: The meeting will facilitate the development of fundamental technologies and the multi-disciplinary applications of proteomics.     

Urine proteome of autosomal dominant polycystic kidney disease patients

Background

Autosomal dominant polycystic kidney disease (ADPKD) is responsible for 10% of cases of the end stage renal disease. Early diagnosis, especially of potential fast progressors would be of benefit for efficient planning of therapy. Urine excreted proteome has become a promising field of the search for marker patterns of renal diseases including ADPKD. Up to now however, only the low molecular weight fraction of ADPKD proteomic fingerprint was studied. The aim of our study was to characterize the higher molecular weight fraction of urinary proteome of ADPKD population in comparison to healthy controls as a part of a general effort aiming at exhaustive characterization of human urine proteome in health and disease, preceding establishment of clinically useful disease marker panel.

Results

We have analyzed the protein composition of urine retentate (>10 kDa cutoff) from 30 ADPKD patients and an appropriate healthy control group by means of a gel-free relative quantitation of a set of more than 1400 proteins. We have identified an ADPKD-characteristic footprint of 155 proteins significantly up- or downrepresented in the urine of ADPKD patients. We have found changes in proteins of complement system, apolipoproteins, serpins, several growth factors in addition to known collagens and extracellular matrix components. For a subset of these proteins we have confirmed the results using an alternative analytical technique.

Conclusions

Obtained results provide basis for further characterization of pathomechanism underlying the observed differences and establishing the proteomic prognostic marker panel.     

脑脊液蛋白质组技术及临床应用研究进展

脑脊液(CSF)围绕并支持中枢神经系统(CNS),包括脑室和蛛网膜下腔,由于脑脊液与中枢神经系统直接接触,所以其是寻找中枢神经系统疾病生物标记物的重要来源。国内外学者开展了大量CSF蛋白质组学的研究工作,并取得了较大进展。文中综述了近年来CSF蛋白质组学技术及临床应用研究进展。     

Comprehensive two-dimensional liquid chromatography mass spectrometric profiling of the rat hippocampal proteome

In this study, we performed the first high‐throughput and comprehensive proteomic profiling of the rat hippocampal proteome. Using a combination of 2‐D LC‐MS and data analysis with the Rosetta Elucidator^® system, we identified 1340 unique proteins. Functional classification showed that most of these were associated with synaptic function and comprised a high proportion of phosphorylated proteins and analytically challenging classes of membrane proteins such as ion channel receptor subunits.     

A comprehensive analysis of Trypanosoma brucei mitochondrial proteome

The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2‐D LC‐MS/MS and gel‐LC‐MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web‐based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.     

Ultrasensitive proteome analysis using paramagnetic bead technology

In order to obtain a systems‐level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity‐limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single‐Pot Solid‐Phase‐enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub‐microgram amounts of material. These data present a first view of developmental stage‐specific proteome dynamics in Drosophila at a single‐embryo resolution, permitting characterization of inter‐individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.     

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2‐D peptide separations employing mixed mode reversed phase anion exchange (MM (RP‐AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP‐AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP‐AX) compared with the traditional SCX hyphenated to RP‐LC‐MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP‐AX) for 2‐D LC proteomic studies was also extended in the analysis of iTRAQ‐labelled HeLa and cyanobacterial proteomes using nano‐flow chromatography interfaced to the MS/MS. We demonstrate MM (RP‐AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.     

Human saliva proteome analysis and disease biomarker discovery

Human saliva is an attractive body fluid for disease diagnosis and prognosis because saliva testing is simple, safe, low-cost and noninvasive. Comprehensive analysis and identification of the proteomic content in human whole and ductal saliva will not only contribute to the understanding of oral health and disease pathogenesis, but also form a foundation for the discovery of saliva protein biomarkers for human disease detection. In this article, we have summarized the proteomic technologies for comprehensive identification of proteins in human whole and ductal saliva. We have also discussed potential quantitative proteomic approaches to the discovery of saliva protein biomarkers for human oral and systemic diseases. With the fast development of mass spectrometry and proteomic technologies, we are enthusiastic that saliva protein biomarkers will be developed for clinical diagnosis and prognosis of human diseases in the future.     

The plasma proteome, adductome and idiosyncratic toxicity in toxicoproteomics research

Toxicoproteomics uses the discovery potential of proteomicsin toxicology research by applying global protein measurementtechnologies to biofluids and tissues after host exposure toinjurious agents. Toxicoproteomic studies thus far have focusedon protein profiling of major organs and biofluids such as liverand blood in preclinical species exposed to model toxicants.The slow pace of discovery for new biomarkers, toxicity signaturesand mechanistic insights is partially due to the limited proteomecoverage derived from analysis of native organs, tissues andbody fluids by traditional proteomic platforms. Improved toxicoproteomicanalysis would result by combining higher data density LC-MS/MSplatforms with stable isotope labelled peptides and paralleluse of complementary platforms. Study designs that remove abundantproteins from biofluids, enrich subcellular structures and includecell specific isolation from heterogeneous tissues would greatlyincrease differential expression capabilities. By leveragingresources from immunology, cell biology and nutrition researchcommunities, toxicoproteomics could make particular contributionsin three inter-related areas to advance mechanistic insightsand biomarker development: the plasma proteome and circulatingmicroparticles, the adductome and idiosyncratic toxicity.      

Multivariate denoising methods combining wavelets and principal component analysis for mass spectrometry data

The identification of new diagnostic or prognostic biomarkers is one of the main aims of clinical cancer research. In recent years, there has been a growing interest in using mass spectrometry for the detection of such biomarkers. The MS signal resulting from MALDI‐TOF measurements is contaminated by different sources of technical variations that can be removed by a prior pre‐processing step. In particular, denoising makes it possible to remove the random noise contained in the signal. Wavelet methodology associated with thresholding is usually used for this purpose. In this study, we adapted two multivariate denoising methods that combine wavelets and PCA to MS data. The objective was to obtain better denoising of the data so as to extract the meaningful proteomic biological information from the raw spectra and reach meaningful clinical conclusions. The proposed methods were evaluated and compared with the classical soft thresholding denoising method using both real and simulated data sets. It was shown that taking into account common structures of the signals by adding a dimension reduction step on approximation coefficients through PCA provided more effective denoising when combined with soft thresholding on detail coefficients.     

Comparative proteome analysis of thalamus in MK-801-treated rats

Two-dimensional gel-electrophoresis in combination with mass spectrometry is a powerful approach to compare protein expression in brain tissues. Using this proteomic approach, and based on the hypothesis that schizophrenia involves hypoglutamergic brain function, alterations in protein levels in the thalamus of rats treated with the N-methyl-D-aspartate (NMDA) receptor antagonist [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801), as compared to saline-treated animals, were assessed in an unbiased fashion. The rats were divided into two groups; group 1 (short-term treated) and group 2 (long-term treated). In group 1, the levels of seven proteins were increased and four proteins reduced. In group 2, the levels of six proteins were reduced. Several of the altered proteins (heat shock proteins 60 and 72, albumin, dihydropyrimidinase related protein-2, aldolase c, and malate dehydrogenase) have previously been connected to schizophrenia. Alterations of other proteins (dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2, guanine deaminase, alpha-enolase, aconitase, ATP-synthase and alpha-internexin), have not, to the best of our knowledge, earlier been implicated in schizophrenia pathology. Our results show the high potential of using proteomic methods for the validation of animal models of schizophrenia and to identify new proteins involved in the pathophysiological mechanisms of schizophrenia.     

Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species

Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.     

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.