Mass spectrometry‐based absolute protein quantification: <scp>PSAQ</scp>™ strategy makes use of “noncanonical” proteotypic peptides

Jaquinod, Michel; Trauchessec, Mathieu; Huillet, Céline; Louwagie, Mathilde; Lebert, Dorothée; Picard, Guillaume; Adrait, Annie; Dupuis, Alain; Garin, Jérôme; Brun, Virginie; Bruley, Christophe

doi:10.1002/pmic.201100538

Cited by 29 publications

(31 citation statements)

References 17 publications

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“…Proteotypic peptides were selected, and the sequence was confirmed to be unique using the BLAST program. In line with Jaquinod et al (28), five cysteine-containing peptides were retained in the transition list. In contrast, methionine-containing peptides were not included.…”

Section: Design Of Srm Assays and Ms/msmentioning

confidence: 99%

Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering

Trauchessec

Jaquinod

Bonvalot

et al. 2014

Molecular & Cellular Proteomics

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Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length 15 N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering. Molecular & Cellular Proteomics

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Section: Design Of Srm Assays and Ms/msmentioning

confidence: 99%

Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering

Trauchessec

Jaquinod

Bonvalot

et al. 2014

Molecular & Cellular Proteomics

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“…Studies on recombinant Tau enabled us to optimize the digestion, chemical modification, and targeted HRMS detection steps. It has to be noted that all methionines were oxidized to methionine sulfone residues in order to render the corresponding peptides readily amenable to absolute quantification [15]. Tryptic digestion of Tau-441 generated a series of peptides (Supplementary Table 1), 18 of which were detectable in pools of CSF used for developing the method (Fig.…”

Section: Selection Of Peptides From Recombinant Tau Proteinsmentioning

confidence: 99%

“…The identification of specific (proteotypic) Tau peptides, and their absolute quantification, was achieved using recombinant protein [15]. In this work, we used the longer Tau (Tau-441) recombinant proteins in its unlabeled and 15N-labeled full-length forms.…”

Section: Selection Of Peptides From Recombinant Tau Proteinsmentioning

confidence: 99%

“…In recent years, the emergence of targeted proteomic approaches by MS has allowed protein quantification in complex biological fluids [14]. In this work, we used a MS based method for the absolute quantification of protein called Protein Standard for Absolute Quantification (PSAQ) standards [15,16]. We relied on the sensitive multiplex peptide detection capability of targeted high-resolution MS (HRMS) on a Q-Orbitrap to follow in parallel 18 peptides covering Tau N-terminus, central core and C-terminus.…”

Section: Introductionmentioning

confidence: 99%

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Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies

Barthélemy

Gabelle

Hirtz

et al. 2016

JAD

115

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Abstract. Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimer's disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Tau's molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tauspecific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear 1 These authors jointly directed this work. N.R. Barthélemy et al. / Tau Cerebrospinal Fluid Molecular Profilepalsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

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“…Peptide sequences containing oxidationsensitive residues, methionine only in this case, were excluded. However, these peptides, which usually ionize well, were kept as secondary choices in case no usable sequences would remain after the selection 47 . Peptides containing potential imide-forming residues (NG), potential deamidating residues (DP) or N-terminal glutamine (Q) were excluded as well.…”

Section: Assessment Of Protein Losses During Gel Extraction Rxrα Wasmentioning

confidence: 99%

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

et al. 2013

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The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPAR and RXR, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from 250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNnA binding model for PPAR based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPAR binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPAR copy number. Originally published at: Simicevic, Jovan; Schmid, Adrien W; Gilardoni, Paola A; Zoller, Benjamin; Raghav, Sunil K; Krier, Irina; Gubelmann, Carinne; Lisacek, Frédérique; Naef, Felix; Moniatte, Marc; Deplancke, Bart (2013). Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nature Methods, 10(6):570-576.

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Mass spectrometry‐based absolute protein quantification: PSAQ™ strategy makes use of “noncanonical” proteotypic peptides

Cited by 29 publications

References 17 publications

Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering

Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

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