Absolute Quantification of Proteins in Solutions and in Polyacrylamide Gels by Mass Spectrometry

Havliš, Jan; Shevchenko, Andrej

doi:10.1021/ac035286f

Cited by 94 publications

(107 citation statements)

References 33 publications

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“…1, left panel) is a core step in AQUIP strategy. Although the efficiency of in-solution digestion normally exceeds 80 -90% and is much higher than the 10 -40% resulting from in-gel digestion (46), the in-gel digestion was still chosen because the target protein, such as ERF110, can be enriched from the total cellular proteins by one-dimensional SDS-PAGE separation under SDS-denaturing condition, and it most likely excludes the possibility of refolding of modifying enzymes like protease, kinase, and phosphatase during fractionation and in-gel digestion. According to the same rationale, the highly purified 15 N-coded recombinant ERF110 protein was also isolated through an identical procedure ( Fig.…”

Section: Substantiation Of the Aquip Results Using The Silia-based Rementioning

confidence: 94%

“…For example, a 4-fold increase in site-specific phosphorylation from 25 to 100% of the total protein may result in a complete different cellular and phenotypic response than that from 1 to 4% (14,39). Because the absolute amount of the phosphorylated isoform is calculated either in moles or grams of cell and tissue, the concentration of a phosphoprotein isoform produced under a number of treatments can be compared directly among various protein extracts (46). The comprehensive statistical analysis of the absolute amount of a protein isoform (e.g.…”

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confidence: 99%

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Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism

Shu

Peng

et al. 2012

Molecular & Cellular Proteomics

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Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P isf ) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent 14 N-coded synthetic peptide standards and 15 N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T isf ) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R aqu ). The P isf was finally determined by integrating the two empirically measured variables using the following equation: P isf ‫؍‬ T isf ⅐ R aqu . The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

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Section: Substantiation Of the Aquip Results Using The Silia-based Rementioning

confidence: 94%

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confidence: 99%

Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism

Shu

Peng

et al. 2012

Molecular & Cellular Proteomics

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“…[5][6][7][8][9][10][11][12][13][14] This enables the simplification of the quantification process to the analysis of short sequences of amino acids which are amenable to standard LC/MS techniques. Using isotopically labeled forms of the peptides as internal standards potentially introduces the advantages of reliability, accuracy, and repeatability into protein quantification that have been demonstrated in the analysis of "small molecules" with isotope dilution mass spectrometry (IDMS).…”

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confidence: 99%

Protein Quantification by Isotope Dilution Mass Spectrometry of Proteolytic Fragments: Cleavage Rate and Accuracy

et al. 2008

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The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U ) 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.The past decade has seen a significant increase in the development of mass spectrometric methods for protein quantification.1,2 Although it is viable to directly analyze intact proteins 3,4 by liquid chromatography/mass spectrometry (LC/MS), most of the examples of quantification that have been described are based on specific cleavage by enzymatic proteolysis (digestion) of the proteins down to smaller fragments, most of which are still long enough in amino acid sequence to provide specificity for the precursor protein, even in a complex mixture. [5][6][7][8][9][10][11][12][13][14] This enables the simplification of the quantification process to the analysis of short sequences of amino acids which are amenable to standard LC/MS techniques. Using isotopically labeled forms of the peptides as internal standards potentially introduces the advantages of reliability, accuracy, and repeatability into protein quantification that have been demonstrat...

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“…Relative and absolute quantification was achieved by titrating SIS peptides into each sample to produce ion signals similar in intensity to those observed for each endogenous peptide. Because of the possibility of incomplete digestion and peptide recovery from the gel, absolute quantification by LC-MRM establishes a minimum value for protein expression (55). However, well-controlled protein digestion and peptide recoveries are highly reproducible enabling comparisons between different samples.…”

Section: Rationale For Selection Of Sds-page Coupled With Lc-mrm-immumentioning

confidence: 99%

“…This choice was based on the prior performance and ease of implementation of SDS-PAGE as a fractionation method prior to liquid chromatography coupled to tandem mass spectrometry peptide sequencing (GeLC-MS/MS), a method that has been extensively used to catalog proteomes (47)(48)(49)(50)(51). Although most of these targets would not be detected in GeLC-MS/MS, the use of LC-MRM with absolute quantification (52)(53)(54)(55) enables quantification of these proteins by comparison to known amounts of stable-isotope-labeled standard (SIS) peptides. In this article, we report the quantitative analysis of signaling pathways and biological processes related to melphalan-resistance in MM.…”

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confidence: 99%

Monitoring a Nuclear Factor-κB Signature of Drug Resistance in Multiple Myeloma

Xiang

Remily-Wood

Oliveira

et al. 2011

Molecular & Cellular Proteomics

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The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-B subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were Multiple myeloma (MM)1 is an incurable malignancy of plasma cells harbored in the bone marrow, which is clinically characterized by secretion of monoclonal antibodies, calcium dysregulation, anemia, lytic bone lesions, and kidney damage. For five decades, therapeutic regimens for MM have included the alkylating agent, melphalan (p-di-2-chloroethylamino-1-phenylalanine, L-phenylalanine mustard, or L-PAM) (1). Therapy with L-PAM (alone or in combination with novel agents) remains the standard of care for transplant-ineligible patients and is the backbone of high-dose therapy associated with autologous stem cell transplant. Although MM patients initially respond to chemotherapy, treatment failure is inevitable because of the emergence of drug resistance. Because of the importance of detecting acquired drug resistance (ADR) for patient assessment and treatment, numerous mechanisms of ADR have been elucidated for MM in vitro, utilizing cell line models developed by chronic exposure to chemotherapy (2-7). These models were designed to identify potential drug targets to reverse ADR as well as to discover prognostic or chemopredictive biomarkers that could be translated to assessment of MM patients. These isogenic model systems clearly show that drug resistance is derived from multiple factors and that prediction of response can not depend on the measurement of one pathway.The biology of one such model of ADR, the 8226/LR5 cell line, selected by chronic exposure of RPMI-8226 MM cells to the DNA-damaging agent, melphalan, has been extensively characterized, leading to the identification of causative mechFrom the ‡Molecular Oncology, §Experimental Therapeutics,

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Absolute Quantification of Proteins in Solutions and in Polyacrylamide Gels by Mass Spectrometry

Cited by 94 publications

References 33 publications

Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism

Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism

Protein Quantification by Isotope Dilution Mass Spectrometry of Proteolytic Fragments: Cleavage Rate and Accuracy

Monitoring a Nuclear Factor-κB Signature of Drug Resistance in Multiple Myeloma

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