Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome.
TL;DR: Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies and allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals.
Abstract: Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC-MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.
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TL;DR: Current methods for target identification of small molecules are summarized, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound.
Abstract: Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins.
384 citations
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TL;DR: The focus of the review has been on glycoanalytical chemistry, which aims to isolate and structurally characterize biologically important glycoconjugates and synthesize carbohydrate structures for biochemical investigations, enabling technologies and medical applications and providing new therapeutics.
Abstract: 1.1. General Considerations
Structural intricacies of carbohydrate molecules and their propensity to form varied linkages, substitutions, and branching patterns have fascinated many generations of chemists, as have the three-dimensional aspects of carbohydrate interactions with other biomolecules. The steadily increasing biochemical knowledge in this area has further added to the increasing importance of the field now referred to as “glycobiology” or, more generally, “glycoscience”. Yet, most of the emphasis over the last 50 years or so has been on two other classes of important biopolymers, namely nucleic acids and proteins. However, in the “post-genomic era”, complex carbohydrates can no longer be neglected, as it is becoming clear to many scientists that most mammalian proteins are glycosylated, and microbial systems and plants can have their own unique monosaccharide building blocks and special ways they can be interconnected and branched into unusual structures. Throughout evolution and the development of living organisms, glycoconjugates must have played major roles, no doubt due to their unusual biological selectivities, which, in turn, could well be due to the enormous information capacity of the “sugar code”.1,2
Throughout the 1980s, the multilateral importance of glycoconjugates in biology and medicine was recognized,3-6 albeit with an understanding that only new methodological approaches and systematic investigations would further define new vistas and provide intimate knowledge of how complex carbohydrates participate in all life processes. Today’s glycoscience is a multidisciplinary undertaking in which chemistry is expected to have an important role to describe the most complex structural aspects of sugars and their conjugates with other biological molecules. While the biological and biomedical relevance of studying glycosylation and sugar–protein and sugar–sugar interactions will undoubtedly be guided by advances in other respective fields (immunology, cancer research, parasitology, cell biology, and developmental biology, among others), the chemical disciplines’ two major tasks are to (a) isolate and structurally characterize biologically important glycoconjugates and (b) synthesize carbohydrate structures for biochemical investigations, enabling technologies and medical applications and providing new therapeutics. While the goals and directions of carbohydrate synthesis have been summarized elsewhere,7-11 the focus of our review has been on glycoanalytical chemistry. The synthetic and bioanalytical directions are not mutually exclusive, as new structural findings will undoubtedly provide further rationale for synthetic efforts and these, in turn, the availability of standards for structural verification.
Since publication of the review on structural investigations of glycoconjugates at high sensitivity12 in these pages a decade ago, the field of analytical glycobiology has seen dramatic changes in its scope and depth. It is widely appreciated within the glycoscience community and increasingly by others that both new techniques and instrumentation and the established (albeit optimized) analytical methodologies have played very important roles in advancing the science of glycoconjugates to its current stage. Due to their different physical and chemical characteristics, the main classes of glycoconjugates, i.e. glycoproteins, glycolipids, polysaccharides, and proteoglycans with their highly charged constituents, glycosaminoglycans, demand somewhat specialized analytical and structural elucidation approaches. Our review will largely be focused on glycoproteins and their associated glycans, hoping that other scientists will describe the analytical aspects of the remaining glycoconjugate biomolecules elsewhere.
The early advances in proteomics, the scientific area mostly preoccupied with identification and structural characterization of proteins, have led to diverse activities in protein post-transitional modifications (PTMs), which are often associated with important biological activities. Glycosylation of proteins is arguably the most widely spread and functionally most intriguing PTM in nature. It is already known that certain glycosylation patterns in proteins give rise to functional variance, with far-reaching consequences for health-disease issues, immunological disorders, toxicity effects, microbial invasion processes, etc. To investigate any of these highly important processes in sufficient molecular detail, analytical techniques capable of a high degree of structural elucidation and measurement sensitivity are currently needed.
Within the plethora of new “-omics fields” (genomics, transcriptomics, lipidomics, metabolomics, etc.), the fields of glycoproteomics and glycomics have started to assume their respectable roles. Analytical glycobiology, representing both glycomics and glycoproteomics, now shares access to new measurement technologies that enable characterization and quantification of molecular processes in living organisms. Extensive glycomic and glycoproteomic data that can nowadays be generated with modern techniques and instrumentation are likely to enrich the “systems biology” approach.13-17 Both fields have started to contribute substantially to a better understanding of multicellular interactions in eukaryotic systems and important issues pertaining to human health and disease.18-23 Additionally, the long-held view that glycosylation is unimportant in prokaryotic systems is no longer defensible.24,25
Since our previous review12 in this journal, much progress has been achieved in terms of methodological developments toward better, more informative, and more sensitive measurements of glycoproteins and their glycan components. In addition, many conceptually important applications of new tools already point to the future needs for dealing with the enormous complexity of glycopeptides and oligosaccharide mixtures extracted from biological tissues and physiological fluids. The relatively recent interest of the pharmaceutical and biotech industries in recombinant glycoproteins, such as monoclonal antibodies, for treatment of cancer and other diseases,26-30 demands the use and further development of glycomic and glycoproteomic analytical procedures as well. Similarly to our previous report,12 the current review has been organized to discuss separately recent advances in glycoproteomics and glycomics, dealing first with the isolation and direct analysis of glycoproteins, followed by the description of advances in glycopeptide analysis and determination of the sites of glycosylation, and moving toward the analysis of complex glycan mixtures. Even more today than 10 years ago, mass spectrometry (MS) is the most prominent methodology in the arsenal of glycoprotein analysis tools. A number of new MS techniques, previously unexplored or insufficiently developed, are now at the center of attention of glycobiologists. At the sensitivity levels required by contemporary glycobiology, MS and tandem MS (MSn) techniques are currently the only means to provide reliable structural information. Carbohydrate derivatization (chemical modification of carbohydrates at microscale) uniquely enables certain MS measurements in terms of enhanced sensitivity and structural information.
Due to the enormous “chemical space” for carbohydrate structural complexity,1,2 MS alone, no matter how sophisticated, is unlikely to provide all needed answers. However, in combinations with modern separation methodologies (different forms of chromatography and electrophoresis) that provide unique component resolution in time and space, MS detection and identification capabilities become enormously enriched. The past decade has seen substantial improvements in the chromatographic analysis of complex carbohydrates: (1) transition from the conventional-scale columns to capillary column dimensions, or even microchips, with the resulting gains in mass sensitivity of measurements; and (2) rapidly increasing use of stable and reliable hydrophilic column materials and graphitized carbon adsorbents. Further advances in capillary chromatographic separations pertain to effective resolution of very complex mixtures as well as the frequently needed separation of different isomers. Chromatographic advances of the recent years also relate to simple purifications of samples (analysis steps now often referred to as solid-phase extraction, or SPE) or the more sophisticated microcolumn lectin or affinity materials needed in group separations and preconcentration of certain glycoproteins for analysis. The past decade has also witnessed a rapid development of glycan array technologies, in which the surface-bound glycan structures (either synthesized or isolated from natural mixtures) are presented to glycan-binding proteins in biological samples.31-33 While these enabling technologies are novel and exciting, they will not be covered in this review, which primarily emphasizes techniques leading to structural elucidation of glycoproteins. Likewise, immunologically based measurements will not be discussed.
266 citations
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TL;DR: A comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry identified 1,823 proteins, of which 671 proteins have not previously been reported as constituents of human urine.
Abstract: The study of the human urinary proteome has the potential to offer significant insights into normal physiology as well as disease pathology. The information obtained from such studies could be applied to the diagnosis of various diseases. The high sensitivity, resolution, and mass accuracy of the latest generation of mass spectrometers provides an opportunity to accurately catalog the proteins present in human urine, including those present at low levels. To this end, we carried out a comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry. Importantly, we used the Orbitrap for detecting ions in both MS (resolution 60 000) and MS/MS (resolution 15 000) modes. To increase the depth of our analysis, we characterized both unfractionated as well as lectin-enriched proteins in our experiments. In all, we identified 1823 proteins with less than 1% false discovery rate, of which 671 proteins have not previously been reported as constitue...
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TL;DR: Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies.
Abstract: The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study inter-individual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called “split by taxonomy id” that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification. Microgram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host–pathogen interactions and clinical saliva diagnostics.
160 citations
Cites background from "Quantitative analysis of the intra-..."
...2b), whereas in plasma and urine they already account for more than 90 % and 58 % of the total, respectively [30, 31]....
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TL;DR: An experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements is presented and the stability of the platform and its suitability for discovery proteomics studies is demonstrated.
Abstract: To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC–MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue d...
157 citations
References
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TL;DR: The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing.
Abstract: Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
35,225 citations
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TL;DR: MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data, detects peaks, isotope clusters and stable amino acid isotope–labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space and achieves mass accuracy in the p.p.b. range.
Abstract: Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
12,340 citations
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TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
6,597 citations
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TL;DR: A method is described, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics and allows single-run analyses of organelles and an unprecedented depth of proteome coverage.
Abstract: A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
6,096 citations
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TL;DR: A novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips, finding that the Stage system is well-suited as a universal sample preparation system for proteomics.
Abstract: Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
2,358 citations