Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

Yocum, Anastasia K.; Chinnaiyan, Arul M.

doi:10.1093/bfgp/eln056

Cited by 109 publications

(82 citation statements)

References 55 publications

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“…Interestingly, the differences in number and type of cleaved proteins in the forward and reverse data sets highlight the potential pitfalls of reverse type experiments. Future studies using multiple reaction monitoring-based quantitation or single cell-based analyses should help to dissect the details of caspase-1 activity by different inflammatory signals (52).…”

Section: Reverse Versus Forward Degradomics Using Subtiligasementioning

confidence: 99%

Inflammatory Stimuli Regulate Caspase Substrate Profiles

Agard

Maltby

Wells

2010

Molecular & Cellular Proteomics

193

152

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The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometrybased proteomics to identify newly cleaved proteins. Analysis of THP-1 monocytic cell lysates treated with recombinant purified caspases identified 82 putative caspase-1 substrates, three putative caspase-4 substrates, and no substrates for caspase-5. By contrast, inflammatory caspases activated in THP-1 cells by mimics of gout (monosodium urate), bacterial infection (lipopolysaccharide and ATP), or viral infection (poly(dA⅐dT)) were found to cleave only 27, 16, and 22 substrates, respectively. Quantitative stable isotope labeling with amino acids in cell culture (SILAC) comparison of these three inflammatory stimuli showed that they induced largely overlapping substrate profiles but different extents of proteolysis. Interestingly, only half of the cleavages found in response to proinflammatory stimuli were contained within our set of 82 in vitro cleavage sites. These data provide the most comprehensive set of caspase-1-cleaved products reported to date and indicate that caspases-4 and -5 have far fewer substrates. Comparisons between the in vitro and in vivo data highlight the importance of localization in regulating inflammatory caspase activity. Finally, our data suggest that inducers of inflammation may subtly alter caspase-1 substrate profiles. Molecular & Cellular Proteomics 9:880 -893, 2010.Proteases are ubiquitous, representing more than 2% of the total human genome, and essential to diverse cellular processes, including catabolism, cell death, and immune function (1-3). Despite their biological importance, the identification of cellular protease substrates remains challenging. Historically, researchers gained insight into protease specificity by assaying enzyme activities against peptide libraries or displays of short peptide sequences on biomolecules (4 -7). More recently, we and others have developed technologies to directly identify protease substrates using differential gel mobilities or by enriching for new protein N termini that result from protein cleavage (8 -12). These proteomics methods have been applied to the widespread proteolysis that occurs during apoptosis to reveal hundreds of novel caspase substrates. Although this is an important demonstration of the power of these technologies, most proteolysis signaling pathways target a far more limited scope of substrates (13). In this study, we extended an enzymatic N-terminal enrichment technique to study the inflammatory caspases, a family of proteases that enact far more limited proteolysis.The human caspases (cysteine aspartyl proteases) are a family of 12 homodimeric cysteine proteases that cleave proteins immediately...

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Section: Reverse Versus Forward Degradomics Using Subtiligasementioning

confidence: 99%

Inflammatory Stimuli Regulate Caspase Substrate Profiles

Agard

Maltby

Wells

2010

Molecular & Cellular Proteomics

193

152

View full text Add to dashboard Cite

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“…In order to utilize triple quadrupole mass spectrometry for protein quantification, prior protein digestion and quantification of peptide products through a bottom-up approach has been the norm [7,24,25]. However, such methods can be time-consuming and possible errors in the protein digestion steps can easily propagate, which can compromise precision and accuracy [26].…”

Section: Introductionmentioning

confidence: 99%

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

Wang

Combe²,

Schug

2016

J. Am. Soc. Mass Spectrom.

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Abstract. Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostatespecific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

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“…With well defined transitions and chromatographic profiling, up to 100 transitions of known proteins could be quantified simultaneously (22). In practice, LC/MS/MS-MRM has been employed to validate biomarkers identified by other high throughput quantitative proteomics (23,24). In the work reported here, we developed an LC/MS/MS-MRM method to relatively quantify multisite modifications of histones simultaneously with high selectivity and up to 5 orders of magnitude of linearity.…”

mentioning

confidence: 99%

A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation

Darwanto

Curtis

Schrag

et al. 2010

Journal of Biological Chemistry

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Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the crosstalk relationship between H2B ubiquitination and H3 Lys-79 methylation.Acetylation, methylation, and ubiquitination are widely known modifications of lysine in histones (1). Histone acetylation normally plays a vital role in gene activation with an exception when acetylation is utilized for other functions such as protein-protein interactions (2, 3). The function of histone methylation depends on the site that is modified, i.e. H3 Lys-9, Lys-27 and H4 Lys-20 methylation are associated with heterochromatin where genes are predominantly in a silenced state, whereas H3 Lys-4 and Lys-79 methylation are linked to euchromatin where the majority of genes are in an active format (4). Methylation may also function oppositely, under two different conditions such that both H3 Lys-9 and Lys-36 methylation have a positive effect in the coding region and negative effect in the promotor region (5, 6). Histone H2B ubiquitination has been demonstrated in vitro to be required for histone H3 Lys-4 and Lys-79 di-and tri-methylation, indicating a cross-talk pathway exists between the two types of modifications in two histone subclasses in the process of regulating gene expression (7-9).Human leukemia cell line U937 typically contains reciprocal CALM-AF10 2 fusion genes resulting from the rearrangement of CALM and AF10 genes (10). These CALM-AF10 and/or AF10/CALM fusion proteins were identified in many leukemia patients with acute myeloid, acute lymphoblastic, and T cell acute lymphoblastic leukemia (11-13). The AF10 protein associates with the histone H3 methyltransferase hDot1L in the nucleus (14, 15). Literally, the fusion protein, CALM-AF10, might compete with the endogenous AF10 protein for hDot1L binding and bring the bound Dot1 out of the nucleus to the cytoplasm along with the shuttling vehicle of CALM. As a consequence, the net concentration of the hDot1L protein in the nucleus decreases, ...

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Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

Cited by 109 publications

References 55 publications

Inflammatory Stimuli Regulate Caspase Substrate Profiles

Inflammatory Stimuli Regulate Caspase Substrate Profiles

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation

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