Proteomic tools for quantitation by mass spectrometry

Lill, Jennie R.

doi:10.1002/mas.10048

Cited by 146 publications

(92 citation statements)

References 63 publications

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“…The amino acids were quantified using ion traces at m/z116. 1 Liquid Chromatography/Mass Spectrometry. Purity checks of the T6*, T12*, and hGH solutions were done on a micrOTOF-Q (Bruker Daltonik GmbH, Bremen, Germany) in direct infusion ESI (positive ion) mode infusing the pertaining solution at 3 µL/ min.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

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

et al. 2008

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“…For optimization studies, four aliquots (72 g protein content each) of an MCF-7 (EMEM/insulin culture) protein extract digest solution, spiked with standards after digestion but before SPEC-PTC18 clean-up (see above), were labeled with iTRAQ reagents 114, 115, 116, and 117, and mixed in a ratio of 0.2:1:1:5. Alternatively, different amounts (4,20,20, and 100 g) of the same cell extract were labeled with iTRAQ reagents and mixed in a ratio of 1:1:1:1, to generate the same final protein ratios of 0.2:1:1:5. In addition, three aliquots (5, 25, and 127 g) of a standard protein mix digest solution (0.5 M) were labeled with iTRAQ reagents 114, 116, and 117, and combined 1:1:1 to generate protein ratios of 0.2:1:5.…”

Section: Itraq Labelingmentioning

confidence: 99%

“…According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13].…”

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confidence: 99%

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Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Armenta

Hoeschele

Lazar

2009

J. Am. Soc. Mass Spectrom.

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An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

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“…Quantitation Reagents; ICAT and ITRAQ Several protocols exist for the relative or absolute quantitation of proteins by mass spectrometry (Lill, 2003;Yan & Chen, 2005). Amongst these protocols are the isotopic labeling tools known as ICAT and ITRAQ reagents that allow parallel multiplex labeling for relative quantitation of protein samples followed by tandem mass spectrometric analysis.…”

Section: Other Recent Proteomic Applicationsmentioning

confidence: 99%

Microwave‐assisted proteomics

Lill¹,

Ingle²,

Liu³

et al. 2007

Mass Spectrometry Reviews

145

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State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instrumentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characterization workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectrometric characterization. Techniques for microwave-assisted biocatalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of posttranslational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied. #

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Proteomic tools for quantitation by mass spectrometry

Cited by 146 publications

References 63 publications

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

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

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Microwave‐assisted proteomics

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