Clinical Proteomics

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.

Annals of the New York Academy of SciencesVolume 956, Issue 1 p. 523-526 Hyperdeviation and Static Ocular Counterroll in Unilateral Abducens Nerve Palsy AGNES M.F. WONG, Corresponding Author AGNES M.F. WONG Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Department of Ophthalmology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaAddress for correspondence: Dr. Agnes Wong, 5-440 WW, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8. Voice: 416-501-5100; fax: 416-603-5596; [email protected].Search for more papers by this authorDOUGLAS TWEED, DOUGLAS TWEED Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Physiology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaSearch for more papers by this authorJAMES A. SHARPE, JAMES A. SHARPE Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Department of Ophthalmology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaSearch for more papers by this author AGNES M.F. WONG, Corresponding Author AGNES M.F. WONG Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Department of Ophthalmology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaAddress for correspondence: Dr. Agnes Wong, 5-440 WW, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8. Voice: 416-501-5100; fax: 416-603-5596; [email protected].Search for more papers by this authorDOUGLAS TWEED, DOUGLAS TWEED Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Physiology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaSearch for more papers by this authorJAMES A. SHARPE, JAMES A. SHARPE Division of Neurology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, Canada Department of Ophthalmology, the University of Toronto, and University Health Network - Toronto Western Hospital, Toronto, Ontario, CanadaSearch for more papers by this author First published: 24 January 2006 https://doi.org/10.1111/j.1749-6632.2002.tb02872.xCitations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume956, Issue1NEUROBIOLOGY OF EYE MOVEMENTS: FROM MOLECULES TO BEHAVIORApril 2002Pages 523-526 RelatedInformation

See related article, pages 992–999 Hypertension affects >20% of the general population, and yet its etiologic basis remains unknown in the vast proportion of those affected.1 Hypertension greatly increases the risk of stroke, myocardial infarction, congestive heart failure and renal dysfunction thus making it an important focus of clinical research. Although pharmacological reductions in blood pressure have been shown to decrease the incidence of these adverse consequences,2 large numbers of hypertensive patients go undiagnosed, undertreated, or are nonresponsive to lifestyle modifications and medical therapy.1 As such, there remains a pressing need for an improved understanding of the mechanisms underlying hypertension. Recent advances in genomic and proteomic analyses have led to the discovery of Mendelian forms (monogenetic traits) of hypertension.3,4 Although rare, these mutations which mainly involve altered renal salt handling, provide a molecular basis for understanding the critical role of the kidney during normal blood pressure regulation.5 By contrast, our understanding of the relative contributions of kidney, heart, CNS, and blood vessel function to blood pressure variations in the general population is complicated by the fact that spontaneous hypertension typically arises as a complex quantitative trait affected by differing combinations of genetic and environmental factors. A number of quantitative trait loci (QTL) associated with hypertension have been identified in both animal disease models and human patients, however the affected gene(s) at these sites have remained elusive owing to the large size and complexity of the regions identified.6 The use of congenic rodent strains to reduce the genomic size of QTLs is an important advance in the field (Reviewed in 7). Indeed, an article in this issue of Circulation Research highlights the potential of …

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Balaji Yegneswaran, University of Toronto, University Health Network, Toronto, ON, Canada; Balaji Yegneswaran, University of Toronto, University Health Network; Peter Liao, University of Toronto, University Health Network; S Vairavanathan, University of Toronto, University Health Network; A Zaki, University of Toronto, University Health Network; F Chung, University of Toronto, University Health Network;

Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283-1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676-682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC-MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit.

A comprehensive approach to protein identification and determination of sites of posttranslational modifications (PTMs) in heavily modified proteins was tested. In this approach, termed "reconstructed molecular mass analysis" (REMMA), the molecular mass distribution of the intact protein is measured first, which reveals the extent and heterogeneity of modifications. Then the protein is digested with one or several enzymes, with peptides separated by reversed-phase HPLC, and analyzed by Fourier transform mass spectrometry (FTMS). Vibrational excitation (collisional or infrared) or electron capture dissociation (ECD) of peptide ions provides protein identification. When a measured peptide molecular mass indicates the possibility of a PTM, vibrational excitation is applied to determine via characteristic losses the type and eventually the structure of the modification, while ECD determines the PTM site. Chromatographic peak analysis continues until full sequence coverage is obtained, after which the molecular mass is reconstructed and compared with the measured value. An agreement indicates that the PTM characterization was complete. This procedure applied to the bovine milk PP3 protein containing 25% modifications by weight yielded all known modifications (five phosphorylations, two O- and one N-glycosylation) as well as the previously unreported NeuNAc-Hex-[NeuNAc]HexNAc group O-linked to Ser60. With the FTMS performance improved, REMMA can serve as the basis for high-throughput, high-sensitivity PTM characterization of biological important proteins, which should speed up the proteomics research.

Specogna, AV; Patten, SB; Hill, MD; University of Calgary, Calgary, AB Background: Previous studies have examined predictors of treatment costs after Spontaneous Intracerebral Hemorrhage (ICH) in various countries and healthcare systems. Currently however, there is no clear understanding of which factors are associated with cost in any Canadian setting and thus no way of understanding how to plan ICH healthcare spending. Methods: We used a retrospective case series study design and adjusted linear regression to investigate if an association existed between the total cost of ICH hospital care in a Canadian health centre and age (years), gender (female vs. male), Charlson Comorbidity Index (0 vs. ≥1), in-hospital mortality (alive vs. dead), and having surgery (no vs. yes). Economic, treatment, and patient data were obtained from administrative sources. Total inflation-adjusted hospital cost per discharge was estimated in Canadian Dollars and log transformed for all analyses. Results: Analyses were performed using 987 consecutive ICH discharges from 1999 to 2008. The total cost of ICH hospital care was highly variable (mean cost per discharge=$25,613.96±$36,116.33; min $446.81 to max $326,275.10). Older age (β=–0.0057; 95% CI: –0.0103 to –0.0011) and death in hospital (β=–0.4859; 95% CI: –0.6357 to –0.3342) were significantly associated with lower total hospital cost per discharge. Whereas Charlson Comorbidity Index of ≥1 (β=0.6489; 95% CI: 0.5063 to 0.7916) and having surgery (β=1.3552; 95% CI: 1.1893 to 1.5210) were significantly associated with higher total hospital cost per discharge. Gender was not significantly associated with cost (β=0.0727; 95% CI: –0.0679 to 0.2134). Conclusions: To our knowledge this is the first study to examine predictors of ICH treatment costs in Canada. In a Canadian health centre, ICH treatment costs were significantly associated with patient age, level of comorbidity, in-hospital mortality, and surgical treatment. This study provides evidence that it may be reasonable to consider …

Large-scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures. Fractionation can involve ion exchange separation coupled to reversed-phase separation or, more recently, two reversed-phase separations performed at different pH values. When multidimensional separations are combined with tandem mass spectrometry for protein identification, the strategy is often referred to as multidimensional protein identification technology (MudPIT). MudPIT has been used in either an automated (online) or manual (offline) format. In this study, we evaluated the performance of different MudPIT strategies by both label-free and tandem mass tag (TMT) isobaric tagging. Our findings revealed that online MudPIT provided more peptide/protein identifications and higher sequence coverage than offline platforms. When employing an off-line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high-pressure device, a 5.3% loss in protein identifications is observed. When off-line fractionated samples are loaded via an autosampler, a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column. Moreover, peptide recovery was significantly lower after offline fractionation than in online fractionation. Signal-to-noise (S/N) ratio, however, was not significantly altered between experimental groups. It is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces. Therefore, the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples.

The field of proteomics is rapidly evolving and has emerged as a routine application in many laboratories. It covers not only the identification of proteins but also the quantification and analysis of post-translational protein modifications. During the last few years a series of summer schools teaching comprehensive knowledge in proteomic research and applied techniques have been held. Various research areas were dealt with by international experts in lectures and workshops. The summer schools were addressed to master's and graduate students as well as young post-docs currently moving into the field of proteomics. Here, we give a report on the third European Summer School “Quantification and Post-translational Modifications of Proteins” held at the monastery in Neustift, Brixen/Bressanone, South Tyrol, Italy from August 2 to 9, 2009.

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

FROM THE DECLINE AND FALL OF PROTEIN CHEMISTRY TO PROTEOMICS

Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.

Several years ago, the editors of MCP1 , concerned with the widely varying standards in articles reporting large scale protein identifications, undertook the task of drafting and adopting guidelines for manuscripts that laid out what it felt (and strengthened by considerable advice and comments received from numerous experts in the field) were the minimum set of requirements for reporting and analyzing this type of data (1). The purpose was to raise awareness to the fact that if articles were severely flawed by incorrect or incomplete information, the field of proteomics was bound to suffer a major loss of credibility.

提出了地震微观前兆预报网络系统设计的一整套详细方案，包括地震模型、基岩传感器网络和数据融合、信号处理技术和网络系统设计。本文介绍网络系统设计方案，包括光纤传感器在地震预报网络中的应用、传感器网络平台的选择和网络设计、地震源定位的相干声阵列处理技术、光数据通信技术。