共查询到20条相似文献,搜索用时 31 毫秒
1.
Hui Wang Tujin Shi Wei-Jun Qian Tao Liu Jacob Kagan Sudhir Srivastava 《Expert review of proteomics》2016,13(1):99-114
Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC–MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC–MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC–MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives. 相似文献
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3.
Slebos RJ Brock JW Winters NF Stuart SR Martinez MA Li M Chambers MC Zimmerman LJ Ham AJ Tabb DL Liebler DC 《Journal of proteome research》2008,7(12):5286-5294
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens. 相似文献
4.
Fu X Gharib SA Green PS Aitken ML Frazer DA Park DR Vaisar T Heinecke JW 《Journal of proteome research》2008,7(3):845-854
Detecting differentially expressed proteins is a key goal of proteomics. We describe a label-free method, the spectral index, for analyzing relative protein abundance in large-scale data sets derived from biological samples by shotgun proteomics. The spectral index is comprised of two biochemically plausible features: relative protein abundance (assessed by spectral counts) and the number of samples within a group with detectable peptides. We combined the spectral index with permutation analysis to establish confidence intervals for assessing differential protein expression in bronchoalveolar lavage fluid from cystic fibrosis and control subjects. Significant differences in protein abundance determined by the spectral index agreed well with independent biochemical measurements. When used to analyze simulated data sets, the spectral index outperformed four other statistical tests (Student's t-test, G-test, Bayesian t-test, and Significance Analysis of Microarrays) by correctly identifying the largest number of differentially expressed proteins. Correspondence analysis and functional annotation analysis indicated that the spectral index improves the identification of enriched proteins corresponding to clinical phenotypes. The spectral index is easily implemented and statistically robust, and its results are readily interpreted graphically. Therefore, it should be useful for biomarker discovery and comparisons of protein expression between normal and disease states. 相似文献
5.
Matiss Ozols Alexander Eckersley Kieran T. Mellody Venkatesh Mallikarjun Stacey Warwood Ronan O'Cualain David Knight Rachel E. B. Watson Christopher E. M. Griffiths Joe Swift Michael J. Sherratt 《Aging cell》2021,20(5)
Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age‐related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification‐associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6β4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery. 相似文献
6.
Antibody‐based proteomics play a very important role in biomarker discovery and validation, facilitating the high‐throughput evaluation of candidate markers. Most proteomics‐driven discovery is nowadays based on the use of MS. MS has many advantages, including its suitability for hypothesis‐free biomarker discovery, since information on protein content of a sample is not required prior to analysis. However, MS presents one main caveat which is the limited sensitivity in complex samples, especially for body fluids, where protein expression covers a huge dynamic range. Antibody‐based technologies remain the main solution to address this challenge since they reach higher sensitivity. In this article, we review the benefits and limitations of antibody‐based proteomics in preclinical and clinical biomarker research for discovery and validation in body fluids and tissue. The combination of antibodies and MS, utilizing the best of both worlds, opens new avenues in biomarker research. 相似文献
7.
Callister SJ Barry RC Adkins JN Johnson ET Qian WJ Webb-Robertson BJ Smith RD Lipton MS 《Journal of proteome research》2006,5(2):277-286
Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias to some degree, assigned ranks among the techniques revealed that for most LC-FTICR-MS analyses linear regression normalization ranked either first or second. However, the lack of a definitive trend among the techniques suggested the need for additional investigation into adapting normalization approaches for label-free proteomics. Nevertheless, this study serves as an important step for evaluating approaches that address systematic biases related to relative quantification and label-free proteomics. 相似文献
8.
《Molecular & cellular proteomics : MCP》2010,9(2):225-241
A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.LC-MS/MS provides the most widely used technology platform for proteomics analyses of purified proteins, simple mixtures, and complex proteomes. In a typical analysis, protein mixtures are proteolytically digested, the peptide digest is fractionated, and the resulting peptide fractions then are analyzed by LC-MS/MS (1, 2). Database searches of the MS/MS spectra yield peptide identifications and, by inference and assembly, protein identifications. Depending on protein sample load and the extent of peptide fractionation used, LC-MS/MS analytical systems can generate from hundreds to thousands of peptide and protein identifications (3). Many variations of LC-MS/MS analytical platforms have been described, and the performance of these systems is influenced by a number of experimental design factors (4).Comparison of data sets obtained by LC-MS/MS analyses provides a means to evaluate the proteomic basis for biologically significant states or phenotypes. For example, data-dependent LC-MS/MS analyses of tumor and normal tissues enabled unbiased discovery of proteins whose expression is enhanced in cancer (5–7). Comparison of data-dependent LC-MS/MS data sets from phosphotyrosine peptides in drug-responsive and -resistant cell lines identified differentially regulated phosphoprotein signaling networks (8, 9). Similarly, activity-based probes and data-dependent LC-MS/MS analysis were used to identify differentially regulated enzymes in normal and tumor tissues (10). All of these approaches assume that the observed differences reflect differences in the proteomic composition of the samples analyzed rather than analytical system variability. The validity of this assumption is difficult to assess because of a lack of objective criteria to assess analytical system performance.The problem of variability poses three practical questions for analysts using LC-MS/MS proteomics platforms. First, is the analytical system performing optimally for the reproducible analysis of complex proteomes? Second, can the sources of suboptimal performance and variability be identified, and can the impact of changes or improvements be evaluated? Third, can system performance metrics provide documentation to support the assessment of proteomic differences between biologically interesting samples?Currently, the most commonly used measure of variability in LC-MS/MS proteomics analyses is the number of confident peptide identifications (11–13). Although consistency in numbers of identifications may indicate repeatability, the numbers do not indicate whether system performance is optimal or which components require optimization. One well characterized source of variability in peptide identifications is the automated sampling of peptide ion signals for acquisition of MS/MS spectra by instrument control software, which results in stochastic sampling of lower abundance peptides (14). Variability certainly also arises from sample preparation methods (e.g. protein extraction and digestion). A largely unexplored source of variability is the performance of the core LC-MS/MS analytical system, which includes the LC system, the MS instrument, and system software. The configuration, tuning, and operation of these system components govern sample injection, chromatography, electrospray ionization, MS signal detection, and sampling for MS/MS analysis. These characteristics all are subject to manipulation by the operator and thus provide means to optimize system performance.Here we describe the development of 46 metrics for evaluating the performance of LC-MS/MS system components. We have implemented a freely available software pipeline that generates these metrics directly from LC-MS/MS data files. We demonstrate their use in characterizing sources of variability in proteomics platforms, both for replicate analyses on a single instrument and in the context of large interlaboratory studies conducted by the National Cancer Institute-supported Clinical Proteomic Technology Assessment for Cancer (CPTAC)1 Network. 相似文献
9.
Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies. 相似文献
10.
Steven E. Schutzer 《Proteomics》2014,14(9):991-993
The proteomics work reported by Smith et al. represents a giant step forward in characterizing the cerebrospinal fluid (CSF) proteome in mouse models of human diseases. Whereas prior studies were limited to analysis of CSF pools, Smith et al. (Proteomics 2014, 14, 1102–1106) base their conclusions on data derived from individual mice, thereby capturing a fuller range of the biological diversity present. These results underscore how far proteomics has come in the past few years, developing into a modern tool with the capacity to remove bottlenecks in the study of neuropsychiatric diseases. Past efforts with mass spectrometry (MS) have been hampered by limitations in access to CSF samples, and small volumes when available. These barriers have been overcome with newer MS platforms and advances in sample preparation. We are far closer than before to producing the production of clinically useful proteomic data for biomarker discovery and for deriving insights into pathogenesis that can lead to more effective treatments for many diseases. 相似文献
11.
The role of pattern in biomarker discovery and clinical diagnosis is examined in its historical context. The use of MS-derived pattern is treated as a logical extension of prior applications of non-MS-derived pattern. Criticisms pertaining to specific technology platforms and analytic methodologies are considered separately from the larger issues of pattern utility and deployment in biomarker discovery. We present a hybrid strategy that marries the desirable attributes of high-information content MS pattern with the capability to obtain identity, and explore the key steps in establishing a data analysis pipeline for pattern-based biomarker discovery. 相似文献
12.
Pavel Bouchal Monika Dvorakova Alexander Scherl Spiros D. Garbis Rudolf Nenutil Borivoj Vojtesek 《Proteomics》2013,13(7):1053-1058
Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed. 相似文献
13.
Perlee L Christiansen J Dondero R Grimwade B Lejnine S Mullenix M Shao W Sorette M Tchernev V Patel D Kingsmore S 《Proteome science》2004,2(1):9-22
BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings. 相似文献
14.
Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC‐MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed. 相似文献
15.
Jin G Zhou X Wang H Zhao H Cui K Zhang XS Chen L Hazen SL Li K Wong ST 《Journal of proteome research》2008,7(9):4013-4021
The mass spectrometry (MS) technology in clinical proteomics is very promising for discovery of new biomarkers for diseases management. To overcome the obstacles of data noises in MS analysis, we proposed a new approach of knowledge-integrated biomarker discovery using data from Major Adverse Cardiac Events (MACE) patients. We first built up a cardiovascular-related network based on protein information coming from protein annotations in Uniprot, protein-protein interaction (PPI), and signal transduction database. Distinct from the previous machine learning methods in MS data processing, we then used statistical methods to discover biomarkers in cardiovascular-related network. Through the tradeoff between known protein information and data noises in mass spectrometry data, we finally could firmly identify those high-confident biomarkers. Most importantly, aided by protein-protein interaction network, that is, cardiovascular-related network, we proposed a new type of biomarkers, that is, network biomarkers, composed of a set of proteins and the interactions among them. The candidate network biomarkers can classify the two groups of patients more accurately than current single ones without consideration of biological molecular interaction. 相似文献
16.
《Molecular & cellular proteomics : MCP》2022,21(11):100416
The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery. 相似文献
17.
Disease biomarkers are predicted to be in low abundance; thus, the most crucial step of biomarker discovery is the efficient fractionation of clinical samples into protein sets that define disease stages and/or predict disease development. For this purpose, we developed a new platform that uses peptide-based size exclusion chromatography (pep-SEC) to quantify disease biomarker candidates. This new platform has many advantages over previously described biomarker profiling platforms, including short run time, high resolution, and good reproducibility, which make it suitable for large-scale analysis. We combined this platform with isotope labeling and label-free methods to identify and quantitate differentially expressed proteins in hepatocellular carcinoma (HCC) tissues. When we combined pep-SEC with a gas phase fractionation method, which broadens precursor ion selection, the protein coverage was significantly increased, which is critical for the global profiling of HCC specimens. Furthermore, pep-SEC-LC-MS/MS analysis enhanced the detection of low-abundance proteins (e.g. insulin receptor substrate 2 and carboxylesterase 1) and glycopeptides in HCC plasma. Thus, our pep-SEC platform is an efficient and versatile pre-fractionation system for the large-scale profiling and quantitation of candidate biomarkers in complex disease proteomes. 相似文献
18.
Yusuke Kawashima Naoyuki Takahashi Mamoru Satoh Tatsuya Saito Sayaka Kado Fumio Nomura Hiroyuki Matsumoto Yoshio Kodera 《Proteomics》2013,13(5):751-755
LC‐ESI/MS/MS‐based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large‐scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05–0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step, we devised a new protocol using Invitrosol (IVS), a commercially available surfactant compatible with ESI‐MS; by dissolving the lyophilized peptides in IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets. 相似文献
19.
Beer LA Tang HY Sriswasdi S Barnhart KT Speicher DW 《Journal of proteome research》2011,10(3):1126-1138
Ectopic pregnancy (EP) and normal intrauterine pregnancy (IUP) serum proteomes were quantitatively compared to systematically identify candidate biomarkers. A 3-D biomarker discovery strategy consisting of abundant protein immunodepletion, SDS gels, LC-MS/MS, and label-free quantitation of MS signal intensities identified 70 candidate biomarkers with differences between groups greater than 2.5-fold. Further statistical analyses of peptide quantities were used to select the most promising 12 biomarkers for further study, which included known EP biomarkers, novel EP biomarkers (ADAM12 and ISM2), and five specific isoforms of the pregnancy specific beta-1-glycoprotein family. Technical replicates showed good reproducibility and protein intensities from the label-free discovery analysis compared favorably with reported abundance levels of several known reference serum proteins over at least 3 orders of magnitude. Similarly, relative abundances of candidate biomarkers from the label-free discovery analysis were consistent with relative abundances from pilot validation assays performed for five of the 12 most promising biomarkers using label-free multiple reaction monitoring of both the patient serum pools used for discovery and the individual samples that constituted these pools. These results demonstrate robust, reproducible, in-depth 3-D serum proteome discovery, and subsequent pilot-scale validation studies can be achieved readily using label-free quantitation strategies. 相似文献
20.
Veenstra TD 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,847(1):3-11
The extraordinary developments made in proteomic technologies in the past decade have enabled investigators to consider designing studies to search for diagnostic and therapeutic biomarkers by scanning complex proteome samples using unbiased methods. The major technology driving these studies is mass spectrometry (MS). The basic premises of most biomarker discovery studies is to use the high data-gathering capabilities of MS to compare biological samples obtained from healthy and disease-afflicted patients and identify proteins that are differentially abundant between the two specimen. To meet the need to compare the abundance of proteins in different samples, a number of quantitative approaches have been developed. In this article, many of these will be described with an emphasis on their advantageous and disadvantageous for the discovery of clinically useful biomarkers. 相似文献