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PubMed2003

Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.

The high-throughput identification and accurate quantification of proteins are essential components of proteomic strategies for studying cellular functions and processes. Techniques that are largely based on stable isotope protein or peptide labeling and automated tandem mass spectrometry are increasingly being applied in quantitative proteomic studies. Over the past year, significant progress has been made toward improving and diversifying these technologies with respect to the methods for stable isotope labeling, process automation and data processing and analysis. Advances in stable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed.

Affinity Labels↗

PubMed2006

Functional and quantitative proteomics using SILAC.

Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.

Amino Acids↗

PubMed2004

Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells.

The p38 mitogen-activated protein kinase (MAPK) is a key mediator of stress, extracellular-, growth factor-, and cytokine-induced signaling, and has been implicated in the development of cancer. Our previous work showed evidence for p38 MAPK activation in a subset of transformed follicular lymphomas (Elenitoba-Johnson et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 7259). We demonstrated that inhibition of p38 MAPK by SB203580 resulted in dose- and time-dependent caspase-3-mediated apoptosis. In order to further elucidate the basis of the cellular effects of SB203580, we have employed a systems biologic approach involving cDNA microarray and quantitative proteomic analysis of transformed follicular lymphoma derived-cells (OCI Ly-1) treated with SB203580. Gene expression profiling revealed differential expression (>/=1.5-fold) of 374 genes/ESTs in cells treated for 3 h and 515 genes/ESTs in cells treated for 21 h. The majority (52% at 3 h and 91% at 21 h) were down-regulated, including genes encoding growth cytokines, transcriptional regulators and cytoskeletal proteins. Quantitative proteomic analysis using ICAT-LC-MS/MS identified 277 differentially expressed proteins at 3 h and 350 proteins at 21 h of treatment with SB203580, the majority of which were also down-regulated. Analysis of functional groups of the differentially expressed proteins implicated components of diverse overlapping pathways including the IL-6/phosphatidylinositol 3-kinase, insulin-like growth factor 2/Ras/Raf, WNT8d/Frizzled, MAPK-activated protein kinase 2, and nuclear factor kappaB. The differential phosphorylation status of selected kinase-active proteins was validated by Western blotting analysis. Our complementary genomic and proteomic approach reveal the global cellular consequences of SB203580 treatment and provide insights into its growth inhibitory effect on transformed follicular lymphoma cells.

Cell Line, Transformed↗

PubMed2006

Quantitative proteomic profiling of pancreatic cancer juice.

Pancreatic juice is an exceptionally rich source of cancer-specific proteins shed from cancerous ductal cells into the pancreatic juice. Quantitative proteomic analysis of the proteins specific to pancreatic cancer juice has not previously been reported. We used isotope-code affinity tag (ICAT) technology and MS/MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls. ICAT technology coupled with MS/MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers. A total of 105 proteins were identified and quantified in the pancreatic juice from a pancreatic cancer patient, of which 30 proteins showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls. Many of these proteins have been externally validated. This is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling, and the study reveals numerous proteins that are shown for the first time to be associated with pancreatic cancer, providing candidates for diagnostic biomarkers. One of the identified proteins, insulin-like growth factor binding protein-2 was further validated by Western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue.

Amino Acid Sequence↗

PubMed2006

Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders.

Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB.

Alzheimer Disease↗

PubMed2003

Stable-isotope dimethyl labeling for quantitative proteomics.

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.

Amination↗

PubMed2006

2DDB - a bioinformatics solution for analysis of quantitative proteomics data.

BACKGROUND: We present 2DDB, a bioinformatics solution for storage, integration and analysis of quantitative proteomics data. As the data complexity and the rate with which it is produced increases in the proteomics field, the need for flexible analysis software increases. RESULTS: 2DDB is based on a core data model describing fundamentals such as experiment description and identified proteins. The extended data models are built on top of the core data model to capture more specific aspects of the data. A number of public databases and bioinformatical tools have been integrated giving the user access to large amounts of relevant data. A statistical and graphical package, R, is used for statistical and graphical analysis. The current implementation handles quantitative data from 2D gel electrophoresis and multidimensional liquid chromatography/mass spectrometry experiments. CONCLUSION: The software has successfully been employed in a number of projects ranging from quantitative liquid-chromatography-mass spectrometry based analysis of transforming growth factor-beta stimulated fi-broblasts to 2D gel electrophoresis/mass spectrometry analysis of biopsies from human cervix. The software is available for download at SourceForge.

Computational Biology↗

PubMed2004

Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells.

Cleavable isotope-coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate-treated murine MC3T3-E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT-labeled samples were combined, proteolytically digested, and the cICAT-derivatized peptides isolated using immobilized avidin chromatography. The cICAT-labeled peptides were resolved into 96 fractions by strong cation-exchange (SCX) liquid chromatography (LC). Analysis of the SCX-LC cICAT peptide fractions by microcapillary reversed-phase LC-tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate-responsive gene and was likewise identified as a phosphate-responsive protein in the current analysis. The 1.76-fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41-fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.

Amino Acid Sequence↗

PubMed2006

Quantitative proteomic analysis of drug-induced changes in mycobacteria.

A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.

Antitubercular Agents↗

PubMed2006

Methods of quantitative proteomics and their application to plant organelle characterization.

Many cell biologists wish to know the subcellular localization of proteins of interest. Proteomics methods have the potential to describe the entire protein content of organelles. However, practical limitations in organelle isolation and analysis of low abundance proteins have meant that organelle proteomics has had, until recently, only limited success. Some examples of quantitative proteomic methods and their use in the study of plant organelle proteomes are discussed here. It is concluded that 2D-difference gel electrophoresis (2D-DIGE) as well as differential isotope tagging strategies coupled to non-gel-based LC-MS are proving useful in this area of research.

Arabidopsis↗

PubMed2006

ProRata: A quantitative proteomics program for accurate protein abundance ratio estimation with confidence interval evaluation.

A profile likelihood algorithm is proposed for quantitative shotgun proteomics to infer the abundance ratios of proteins from the abundance ratios of isotopically labeled peptides derived from proteolysis. Previously, we have shown that the estimation variability and bias of peptide abundance ratios can be predicted from their profile signal-to-noise ratios. Given multiple quantified peptides for a protein, the profile likelihood algorithm probabilistically weighs the peptide abundance ratios by their inferred estimation variability, accounts for their expected estimation bias, and suppresses contribution from outliers. This algorithm yields maximum likelihood point estimation and profile likelihood confidence interval estimation of protein abundance ratios. This point estimator is more accurate than an estimator based on the average of peptide abundance ratios. The confidence interval estimation provides an "error bar" for each protein abundance ratio that reflects its estimation precision and statistical uncertainty. The accuracy of the point estimation and the precision and confidence level of the interval estimation were benchmarked with standard mixtures of isotopically labeled proteomes. The profile likelihood algorithm was integrated into a quantitative proteomics program, called ProRata, freely available at www.MSProRata.org.

Algorithms↗

PubMedDate not supplied

Proteolytic 18O-labeling strategies for quantitative proteomics.

A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as "proteolytic 18O-labeling." This technique utilizes a protease and H(2)18O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein's expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions. The present review discusses various aspects of the 18O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications.

Gene Expression Profiling↗

PubMed2005

Quantitative proteomic analysis of mitochondria from primary neuron cultures treated with amyloid beta peptide.

Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of neuron degeneration in Alzheimer's disease (AD). Although several studies have examined the effect of amyloid beta peptide (Abeta), on activities of individual proteins in primary neuron cultures, there have been no studies of the effects of Abeta on the mitochondrial proteome. Here, we quantitatively measured changes in mitochondrial proteins of primary rat cortical neuron cultures exposed to 25 microM Abeta(25-35) for 16 h using isotope coded affinity tag (ICAT) labeling and 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) which allows simultaneous identification and quantification of cysteine-containing proteins. The analysis of enriched mitochondrial fractions identified 10 proteins including sodium/potassium-transporting ATPase, cofilin, dihydropyrimidinase, pyruvate kinase and voltage dependent anion channel 1 that were statistically significantly (P < 0.05) altered in Abeta-treated cultures. Elevations of proteins associated with energy production suggest that cells undergoing Abeta-mediated apoptosis increase synthesis of proteins essential for ATP production and efflux in an attempt to maintain metabolic function.

Amyloid beta-Peptides↗

PubMed2025

T cell population size control by coronin 1 uncovered: from a spot identified by two-dimensional gel electrophoresis to quantitative proteomics.

INTRODUCTION: Recent work identified members of the evolutionarily conserved coronin protein family as key regulators of cell population size. This work originated ~25 years ago through the identification, by two-dimensional gel electrophoresis, of coronin 1 as a host protein involved in the virulence of Mycobacterium tuberculosis. We here describe the journey from a spot on a 2D gel to the recent realization that coronin proteins represent key controllers of eukaryotic cell population sizes, using ever more sophisticated proteomic techniques. AREAS COVERED: We discuss the value of 'old school' proteomics using relatively simple and cost-effective technologies that allowed to gain insights into subcellular proteomes and describe how label-free quantitative (phospho)proteomics using mass spectrometry allowed to disentangle the role for coronin 1 in eukaryotic cell population size control. Finally, we mention potential implications of coronin-mediated cell population size control for health and disease. EXPERT OPINION: Proteome analysis has been revolutionized by the advent of modern-day mass spectrometers and is indispensable for a better understanding of biology. Here, we discuss how careful dissection of physio-pathological processes by a combination of proteomics, genomics, biochemistry and cell biology may allow to zoom in on the unexplored, thereby possibly tackling hitherto unasked questions and defining novel mechanisms.

Proteomics↗

PubMed2001

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.

Affinity Labels↗

PubMed2007

Protein markers of ischemic insult in brain endothelial cells identified using 2D gel electrophoresis and ICAT-based quantitative proteomics.

The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes that lead to the disruption of the BBB, development of vascular inflammation, and subsequent neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two complementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) and isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and quantitative comparison between the two proteomic methods applied to the same experimental system with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and was more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE. However, positive identification of proteins by MS/MS was more reliably done using a 2DE-based method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle, and apoptosis were observed. The functional significance of observed protein changes was evaluated through a multifaceted protein classification and validation process, which included literature mining and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The comparative analyses of protein changes between the in vitro and in vivo models demonstrated a significant correlative relationship, emphasizing the 'translational' value of in vitro endothelial models in neurovascular research.

Aldehyde Oxidoreductases↗

PubMed2003

Quantitative proteomics using mass spectrometry.

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.

Isotopes↗

PubMed2026

Quantitative Proteomic Profiling of Pinctada fucata Shell Nacre Defines a Solubility-Based Type Classification of Shell Matrix Proteins.

Shell matrix proteins (SMPs) are key organic components of molluscan biominerals, yet previous nacre proteomics have remained largely qualitative, limiting evaluation of the abundance and fraction association of individual SMPs. Here, we established a quantitative proteomic approach for the nacreous layer of the pearl oyster Pinctada fucata by integrating optimized shell preservation, stepwise fractionation, and data-independent acquisition (DIA) proteomics. SMPs were separated into an ethylenediaminetetraacetic acid (EDTA)-soluble matrix (ESM), an EDTA-insoluble but sodium dodecyl sulfate/dithiothreitol (SDS/DTT)-soluble matrix (SSM), and an SDS/DTT-insoluble matrix (ISM). DIA outperformed data-dependent acquisition in proteome coverage and enabled quantification of 327 SMPs across a broad dynamic range. Fraction-resolved abundance profiling showed that each fraction was characterized by distinct SMP compositions. To summarize these distributions, we introduced a solubility-based type classification that grouped SMPs into four types according to their quantitative partitioning among fractions. Well-known SMPs, including nacrein, Pif 80, and MSI60, were assigned to intuitively consistent types, whereas proteases, protease inhibitors, and tyrosinases also showed biased type distributions. These results support a three-compartment model of the nacreous layer consisting of (i) an insoluble interlamellar membrane core, (ii) a relatively extractable interfacial layer, and (iii) a soluble matrix fraction enriched in proteins potentially involved in ionic regulation and protein maturation. This study provides a quantitative framework for understanding coordinated SMP functions during nacre formation and for comparative analyses of molluscan shell proteomes.

Animals↗