Global Protein Identification and Quantification Technology Using Two-Dimensional Liquid Chromatography Nanospray Mass Spectrometry

Chelius, Dirk; Zhang, Terry; Wang, Guanghui; Shen, Rong-Fong

doi:10.1021/ac034607k

Cited by 108 publications

(87 citation statements)

References 18 publications

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“…This observation is compatible with reports that show that peak areas (without the need for isotopic or any other type of labeling) generated during LC-MS and 2D LC-MS experiments can be used for relative quantitation of proteins present in related cell lysates (15) and subcellular fractions (13). We have also observed previously that mass spectrometric signal intensities from direct LC-MS runs correlate with the quantitative information obtained through the use of the 2D gel electrophoresis and ICAT methods (23).…”

Section: Quantification Of Protein-derived Peptides By Lc-ms Using Ansupporting

confidence: 91%

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Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a nonisotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase C␥2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospho- Reversible covalent modification of proteins is a key molecular mechanism by which membrane receptor activation is converted into intracellular signaling cascades that mediate physiological change. Engagement of ligands with their receptors results in rapid and transient phosphorylation of downstream protein targets, which as a consequence modify their catalytic properties, cellular localization, and/or binding partners. As a consequence, intracellular signaling pathways can control critical aspects of metabolism, cell cycle progression, migration, differentiation, and protein synthesis. Many diseases are a direct consequence of deregulation in these processes (1, 2); these changes may be detectable by quantitative phosphoprotein analysis.The assessment of the phosphorylation status of specific proteins is commonly performed by immunochemical methods using antisera raised against a peptide containing the site of phosphorylation of the target protein. This approach is restricted to the study of known phosphorylation sites and cannot be used to monitor and discover novel phosphorylated proteins and their sites of phosphorylation. To overcome this proble...

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Section: Quantification Of Protein-derived Peptides By Lc-ms Using Ansupporting

confidence: 91%

“…MS-based quantification approaches that do not depend on protein derivatization or isotopic labeling have also been reported (13)(14)(15). These methods have used the information that is inherent in the integrated peak areas that are generated in LC-MS and two-dimensional (2D) LC-MS experiments, which are in principle proportional to protein abundance.…”

mentioning

confidence: 99%

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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“…Most MS methods for the quantitation of protein phosphorylation assume that MS is not quantitatively reproducible. However, there is increasing evidence that MS can provide some quantitation of protein abundances (22)(23)(24) and protein modifications (25,26) based on the ion signal intensities (ion current). These experiments show that appropriate normalization procedures are essential for deriving quantitative information without the use of stable isotopes.…”

Section: Resultsmentioning

confidence: 99%

Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

Steen

Jebanathirajah

Springer

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

197

177

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Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate͞expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization͞detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.O ne of the most common and important posttranslational protein modification (PTM) is protein phosphorylation (1). It is estimated that Ϸ30% of all proteins in mammalian cells are phosphorylated at any given time and Ϸ5% of a vertebrate genome encodes protein kinases and phosphatases (2), underscoring the importance of this PTM. The presence of various protein kinases and phosphatases permits the use of quickly reversible phosphorylation in a vast number of different, highly regulated pathways and functions, including signal transduction, cell division, and cell differentiation.Knowledge of the phosphorylation site is crucial to a detailed understanding of regulatory processes in cells; this knowledge requires sensitive-analysis methods. Theoretically, the most sensitive methods for the detection of phosphorylation incorporate radioactive phosphorus isotopes before phosphopeptide mapping and͞or Edman degradation (3). However, the incorporation of radioactive isotopes is not possible (e.g., in tissue samples) or is very inefficient in the case of cell culture because of the presence of endogenous unlabeled ATP. Also, high levels of radioactive phosphate incorporation cause cellular damage and, thereby, can alter pho...

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“…In the protein extraction and purification process, SDS was avoided and salt usage was kept low. The in-solution trypsin digest of the BS and M proteomes followed a procedure adapted from Chelius et al (2003). Briefly, purified BS and M stromal proteomes were reduced with DTT and alkylated using iodoacetamide, followed by in-solution digestion by trypsin.…”

Section: Comparative Proteomics By Parallel Ion Chromatograms From Onmentioning

confidence: 99%

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

et al. 2005

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Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway.

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Global Protein Identification and Quantification Technology Using Two-Dimensional Liquid Chromatography Nanospray Mass Spectrometry

Cited by 108 publications

References 18 publications

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

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