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1.
液质联用多反应监测法定量目标多肽或蛋白质 总被引:2,自引:0,他引:2
为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 (MRM)分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析. 相似文献
2.
Mueller LN Rinner O Schmidt A Letarte S Bodenmiller B Brusniak MY Vitek O Aebersold R Müller M 《Proteomics》2007,7(19):3470-3480
Label-free quantification of high mass resolution LC-MS data has emerged as a promising technology for proteome analysis. Computational methods are required for the accurate extraction of peptide signals from LC-MS data and the tracking of these features across the measurements of different samples. We present here an open source software tool, SuperHirn, that comprises a set of modules to process LC-MS data acquired on a high resolution mass spectrometer. The program includes newly developed functionalities to analyze LC-MS data such as feature extraction and quantification, LC-MS similarity analysis, LC-MS alignment of multiple datasets, and intensity normalization. These program routines extract profiles of measured features and comprise tools for clustering and classification analysis of the profiles. SuperHirn was applied in an MS1-based profiling approach to a benchmark LC-MS dataset of complex protein mixtures with defined concentration changes. We show that the program automatically detects profiling trends in an unsupervised manner and is able to associate proteins to their correct theoretical dilution profile. 相似文献
3.
4.
Background
Relative isotope abundance quantification, which can be used for peptide identification and differential peptide quantification, plays an important role in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. However, several major issues exist in the relative isotopic quantification of peptides on time-of-flight (TOF) instruments: LC peak boundary detection, thermal noise suppression, interference removal and mass drift correction. We propose to use the Maximum Ratio Combining (MRC) method to extract MS signal templates for interference detection/removal and LC peak boundary detection. In our method, MRCQuant, MS templates are extracted directly from experimental values, and the mass drift in each LC-MS run is automatically captured and compensated. We compared the quantification accuracy of MRCQuant to that of another representative LC-MS quantification algorithm (msInspect) using datasets downloaded from a public data repository. 相似文献5.
A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers. 相似文献
6.
Kaplan A Söderström M Fenyö D Nilsson A Fälth M Sköld K Svensson M Pettersen H Lindqvist S Svenningsson P Andrén PE Björkesten L 《Journal of proteome research》2007,6(7):2888-2895
Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms. 相似文献
7.
Schmidt A Gehlenborg N Bodenmiller B Mueller LN Campbell D Mueller M Aebersold R Domon B 《Molecular & cellular proteomics : MCP》2008,7(11):2138-2150
LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mixtures. For very complex samples such as complete proteomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abundance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detectable peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. The selected features or a subset thereof are subsequently sequenced in sequential, non-redundant directed LC-MS/MS experiments, and the MS/MS data are mapped back to the original LC-MS feature map in a fully automated manner. The strategy, implemented on an LTQ-FT MS platform, allowed the specific sequencing of 2,000 features per analysis and enabled the identification of more than 1,600 phosphorylation sites using a single reversed phase separation dimension without the need for time-consuming prefractionation steps. Compared with conventional DDA LC-MS/MS experiments, a substantially higher number of peptides could be identified from a sample, and this increase was more pronounced for low intensity precursor ions. 相似文献
8.
Qian Dong Yuxue Liang Xinjian Yan Sanford P. Markey Yuri A. Mirokhin Dmitrii V. Tchekhovskoi 《MABS-AUSTIN》2018,10(3):354-369
We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins. 相似文献
9.
Vissers JP Pons S Hulin A Tissier R Berdeaux A Connolly JB Langridge JI Geromanos SJ Ghaleh B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2009,877(13):1317-1326
An LC-MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC-MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC-MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins. 相似文献
10.
Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes. 相似文献
11.
Recently, many software tools have been developed to perform quantification in LC-MS analyses. However, most of them are specific to either a quantification strategy (e.g. label-free or isotopic labelling) or a mass-spectrometry system (e.g. high or low resolution). In this context, we have developed MassChroQ (Mass Chromatogram Quantification), a versatile software that performs LC-MS data alignment and peptide quantification by peak area integration on extracted ion chromatograms. MassChroQ is suitable for quantification with or without labelling and is not limited to high-resolution systems. Peptides of interest (for example all the identified peptides) can be determined automatically, or manually by providing targeted m/z and retention time values. It can handle large experiments that include protein or peptide fractionation (as SDS-PAGE, 2-D LC). It is fully configurable. Every processing step is traceable, the produced data are in open standard formats and its modularity allows easy integration into proteomic pipelines. The output results are ready for use in statistical analyses. Evaluation of MassChroQ on complex label-free data obtained from low and high-resolution mass spectrometers showed low CVs for technical reproducibility (1.4%) and high coefficients of correlation to protein quantity (0.98). MassChroQ is freely available under the GNU General Public Licence v3.0 at http://pappso.inra.fr/bioinfo/masschroq/. 相似文献
12.
Selected reaction monitoring (SRM) is an accurate quantitative technique, typically used for small-molecule mass spectrometry (MS). SRM has emerged as an important technique for targeted and hypothesis-driven proteomic research, and is becoming the reference method for protein quantification in complex biological samples. SRM offers high selectivity, a lower limit of detection and improved reproducibility, compared to conventional shot-gun-based tandem MS (LC-MS/MS) methods. Unlike LC-MS/MS, which requires computationally intensive informatic postanalysis, SRM requires preacquisition bioinformatic analysis to determine proteotypic peptides and optimal transitions to uniquely identify and to accurately quantitate proteins of interest. Extensive arrays of bioinformatics software tools, both web-based and stand-alone, have been published to assist researchers to determine optimal peptides and transition sets. The transitions are oftentimes selected based on preferred precursor charge state, peptide molecular weight, hydrophobicity, fragmentation pattern at a given collision energy (CE), and instrumentation chosen. Validation of the selected transitions for each peptide is critical since peptide performance varies depending on the mass spectrometer used. In this review, we provide an overview of open source and commercial bioinformatic tools for analyzing LC-MS data acquired by SRM. 相似文献
13.
There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics. 相似文献
14.
Daly DS Anderson KK Panisko EA Purvine SO Fang R Monroe ME Baker SE 《Journal of proteome research》2008,7(3):1209-1217
Comparing a protein's concentrations across two or more treatments is the focus of many proteomics studies. A frequent source of measurements for these comparisons is a mass spectrometry (MS) analysis of a protein's peptide ions separated by liquid chromatography (LC) following its enzymatic digestion. Alas, LC-MS identification and quantification of equimolar peptides can vary significantly due to their unequal digestion, separation, and ionization. This unequal measurability of peptides, the largest source of LC-MS nuisance variation, stymies confident comparison of a protein's concentration across treatments. Our objective is to introduce a mixed-effects statistical model for comparative LC-MS proteomics studies. We describe LC-MS peptide abundance with a linear model featuring pivotal terms that account for unequal peptide LC-MS measurability. We advance fitting this model to an often incomplete LC-MS data set with REstricted Maximum Likelihood (REML) estimation, producing estimates of model goodness-of-fit, treatment effects, standard errors, confidence intervals, and protein relative concentrations. We illustrate the model with an experiment featuring a known dilution series of a filamentous ascomycete fungus Trichoderma reesei protein mixture. For 781 of the 1546 T. reesei proteins with sufficient data coverage, the fitted mixed-effects models capably described the LC-MS measurements. The LC-MS measurability terms effectively accounted for this major source of uncertainty. Ninety percent of the relative concentration estimates were within 0.5-fold of the true relative concentrations. Akin to the common ratio method, this model also produced biased estimates, albeit less biased. Bias decreased significantly, both absolutely and relative to the ratio method, as the number of observed peptides per protein increased. Mixed-effects statistical modeling offers a flexible, well-established methodology for comparative proteomics studies integrating common experimental designs with LC-MS sample processing plans. It favorably accounts for the unequal LC-MS measurability of peptides and produces informative quantitative comparisons of a protein's concentration across treatments with objective measures of uncertainties. 相似文献
15.
Lowenthal MS Gasca-Aragon H Schiel JE Dodder NG Bunk DM 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(26):2726-2732
A mass spectrometry-based antibody selection procedure was developed to evaluate optimal 'capture' monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K(d)) were determined for 'bound mAb-antigen' vs. 'unbound antigen' using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal 'capture' mAbs were determined through evaluation of relative K(d) constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K(d) constants as determined using NIST Standard Reference Material (SRM) 2921 - Human Cardiac Troponin Complex. This ID LC-MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification. 相似文献
16.
Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. 相似文献
17.
Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs ≤ 30% across 10 technical replicates, and at least 93.6% (up to 2,013) had R(2) ≥ 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification. 相似文献
18.
van den Broek I Sparidans RW Schellens JH Beijnen JH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,872(1-2):1-22
With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded. 相似文献
19.
Wang P Tang H Fitzgibbon MP McIntosh M Coram M Zhang H Yi E Aebersold R 《Biostatistics (Oxford, England)》2007,8(2):357-367
Integrated liquid-chromatography mass-spectrometry (LC-MS) is becoming a widely used approach for quantifying the protein composition of complex samples. The output of the LC-MS system measures the intensity of a peptide with a specific mass-charge ratio and retention time. In the last few years, this technology has been used to compare complex biological samples across multiple conditions. One challenge for comparative proteomic profiling with LC-MS is to match corresponding peptide features from different experiments. In this paper, we propose a new method--Peptide Element Alignment (PETAL) that uses raw spectrum data and detected peak to simultaneously align features from multiple LC-MS experiments. PETAL creates spectrum elements, each of which represents the mass spectrum of a single peptide in a single scan. Peptides detected in different LC-MS data are aligned if they can be represented by the same elements. By considering each peptide separately, PETAL enjoys greater flexibility than time warping methods. While most existing methods process multiple data sets by sequentially aligning each data set to an arbitrarily chosen template data set, PETAL treats all experiments symmetrically and can analyze all experiments simultaneously. We illustrate the performance of PETAL on example data sets. 相似文献
20.
Asara JM Zhang X Zheng B Christofk HH Wu N Cantley LC 《Journal of proteome research》2006,5(1):155-163
Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification. 相似文献