Technology, Cambridge, MA Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. W... more Technology, Cambridge, MA Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. White2, Bhavin Patel3, Sebastien Gallien4, Romain Huguet1, Graeme McAlister1, Derek Bailey1, Shannon Eliuk1, Markus Kellmann5, Tabiwang N. Arrey5, Alexander Harder5, Daniel Lopez-Ferrer1, Andreas Huhmer1. Thermo Fisher Scientific, San Jose1, CA, USA; Rockford3, IL, USA; PMSC4, Cambridge, MA, USA; Bremen, Germany5; Department of Biological Engineering2, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA
Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysre... more Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accur...
Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwid... more Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrated robust, sensitive, and reproducible data generation across eleven sites in nine countries on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digiti...
The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium d... more The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans , which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ 1 -dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ 1 -desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and meth...
The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based p... more The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measu... more The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measurement and fast acquisition, have enabled new approaches for targeted quantitative proteomics. More specifically, analyses performed on quadrupole-orbitrap mass spectrometers operated in parallel reaction monitoring (PRM) mode leverage the intrinsic high resolving power and trapping capabilities. The PRM technique offers unmatched degrees of selectivity and analytical sensitivity, typically required to analyze peptides in complex samples, such as those encountered in biomedical research or clinical studies. The features of PRM have provoked a paradigm change in targeted experiments, by decoupling acquisition and data processing. It has resulted in a new analytical workflow comprising distinct methods for each step, thus enabling much larger flexibility. The PRM technique was further enhanced by a new data acquisition scheme, allowing dynamic parameter settings. The potential of the techn...
The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based p... more The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
Quantitative proteomics has benefited from the recent development of mass spectrometers capable o... more Quantitative proteomics has benefited from the recent development of mass spectrometers capable of high-resolution and accurate-mass (HR/AM) measurements. While targeted experiments are routinely performed on triple quadrupole instruments in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode, the quadrupole-orbitrap mass spectrometers allow quantification in MS/MS mode, also known as parallel reaction monitoring (PRM). This technique is characterized by higher selectivity and better confidence in the assignment of the precursor and fragment ions, and thus translates into an improved analytical performance. More fundamentally, PRM introduces a change of the overall paradigm of targeted experiments, by the decoupling of the acquisition and data processing. They rely on two distinct steps, with a simplified acquisition method in conjunction with a flexible, iterative, post-acquisition data processing. This account describes in detail the differ...
Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastica... more Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots. Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectric focusing IEF/SDS-PAGE and 2D Blue native BN/SDS-PAGE was studied. The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H2O2-producing and consuming peroxidase activit...
In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with und... more In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with undetected frameshifts and in-frame stop codons. These interrupted coding sequences (ICDSs) may really be present in the organism or may result from misannotation based on sequencing errors. The reality or otherwise of these sequences has major implications for all subsequent functional characterization steps, including module prediction, comparative genomics and high-throughput proteomic projects. We show here, using Mycobacterium smegmatis as a model species, that a significant proportion of these ICDSs result from sequencing errors. We used a resequencing procedure and mass spectrometry analysis to determine the nature of a number of ICDSs in this organism. We found that 28 of the 73 ICDSs investigated correspond to sequencing errors. The correction of these errors results in modification of the predicted amino acid sequences of the corresponding proteins and changes in annotation. We su...
Molecular & cellular proteomics : MCP, Jan 9, 2015
Targeted high resolution and accurate mass analyses performed on fast sequencing mass spectromete... more Targeted high resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared to the reference method of selected reaction monitoring performed on triple quadrupole instrument. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data a...
MS-based approaches using targeted methods have been widely adopted by the proteomics community t... more MS-based approaches using targeted methods have been widely adopted by the proteomics community to study clinical questions such as the evaluation of biomarkers. At present, the most widely used targeted MS method is the SRM technique typically performed on a triple quadrupole instrument. However, the high analytical demands for performing clinical studies in combination with the extreme complexity of the samples involved are a serious challenge. The segmentation of the biomarker evaluation workflow has only partially alleviated these issues by differently balancing the analytical requirements and throughput at different stages of the process. The recent introduction of targeted high-resolution and accurate-mass analyses on fast sequencing mass spectrometers operated in parallel reaction monitoring (PRM) mode offers new avenues to conduct clinical studies and thus overcome some of the limitations of the triple quadrupole instrument. This article discusses the attributes and specificities of the PRM technique, in terms of experimental design, execution, and data analysis, and the implications for biomarker evaluation. The benefits of PRM on data quality and the impact on the consistency of results are highlighted and the definitive progress on the overall output of clinical studies, including high throughput, is discussed.
High resolution/accurate mass hybrid mass spectrometers have considerably advanced shotgun proteo... more High resolution/accurate mass hybrid mass spectrometers have considerably advanced shotgun proteomics and the recent introduction of fast sequencing capabilities has expanded its use for targeted approaches. More specifically, the quadrupole-orbitrap instrument has a unique configuration and its new features enable a wide range of experiments. An overview of the analytical capabilities of this instrument is presented, with a focus on its application to quantitative analyses. The high resolution, the trapping capability and the versatility of the instrument have allowed quantitative proteomic workflows to be redefined and new data acquisition schemes to be developed. The initial proteomic applications have shown an improvement of the analytical performance. However, as quantification relies on ion trapping, instead of ion beam, further refinement of the technique can be expected.
Technology, Cambridge, MA Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. W... more Technology, Cambridge, MA Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. White2, Bhavin Patel3, Sebastien Gallien4, Romain Huguet1, Graeme McAlister1, Derek Bailey1, Shannon Eliuk1, Markus Kellmann5, Tabiwang N. Arrey5, Alexander Harder5, Daniel Lopez-Ferrer1, Andreas Huhmer1. Thermo Fisher Scientific, San Jose1, CA, USA; Rockford3, IL, USA; PMSC4, Cambridge, MA, USA; Bremen, Germany5; Department of Biological Engineering2, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA
Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysre... more Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accur...
Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwid... more Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrated robust, sensitive, and reproducible data generation across eleven sites in nine countries on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digiti...
The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium d... more The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans , which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ 1 -dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ 1 -desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and meth...
The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based p... more The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measu... more The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measurement and fast acquisition, have enabled new approaches for targeted quantitative proteomics. More specifically, analyses performed on quadrupole-orbitrap mass spectrometers operated in parallel reaction monitoring (PRM) mode leverage the intrinsic high resolving power and trapping capabilities. The PRM technique offers unmatched degrees of selectivity and analytical sensitivity, typically required to analyze peptides in complex samples, such as those encountered in biomedical research or clinical studies. The features of PRM have provoked a paradigm change in targeted experiments, by decoupling acquisition and data processing. It has resulted in a new analytical workflow comprising distinct methods for each step, thus enabling much larger flexibility. The PRM technique was further enhanced by a new data acquisition scheme, allowing dynamic parameter settings. The potential of the techn...
The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based p... more The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.
Quantitative proteomics has benefited from the recent development of mass spectrometers capable o... more Quantitative proteomics has benefited from the recent development of mass spectrometers capable of high-resolution and accurate-mass (HR/AM) measurements. While targeted experiments are routinely performed on triple quadrupole instruments in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode, the quadrupole-orbitrap mass spectrometers allow quantification in MS/MS mode, also known as parallel reaction monitoring (PRM). This technique is characterized by higher selectivity and better confidence in the assignment of the precursor and fragment ions, and thus translates into an improved analytical performance. More fundamentally, PRM introduces a change of the overall paradigm of targeted experiments, by the decoupling of the acquisition and data processing. They rely on two distinct steps, with a simplified acquisition method in conjunction with a flexible, iterative, post-acquisition data processing. This account describes in detail the differ...
Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastica... more Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots. Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectric focusing IEF/SDS-PAGE and 2D Blue native BN/SDS-PAGE was studied. The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H2O2-producing and consuming peroxidase activit...
In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with und... more In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with undetected frameshifts and in-frame stop codons. These interrupted coding sequences (ICDSs) may really be present in the organism or may result from misannotation based on sequencing errors. The reality or otherwise of these sequences has major implications for all subsequent functional characterization steps, including module prediction, comparative genomics and high-throughput proteomic projects. We show here, using Mycobacterium smegmatis as a model species, that a significant proportion of these ICDSs result from sequencing errors. We used a resequencing procedure and mass spectrometry analysis to determine the nature of a number of ICDSs in this organism. We found that 28 of the 73 ICDSs investigated correspond to sequencing errors. The correction of these errors results in modification of the predicted amino acid sequences of the corresponding proteins and changes in annotation. We su...
Molecular & cellular proteomics : MCP, Jan 9, 2015
Targeted high resolution and accurate mass analyses performed on fast sequencing mass spectromete... more Targeted high resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared to the reference method of selected reaction monitoring performed on triple quadrupole instrument. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data a...
MS-based approaches using targeted methods have been widely adopted by the proteomics community t... more MS-based approaches using targeted methods have been widely adopted by the proteomics community to study clinical questions such as the evaluation of biomarkers. At present, the most widely used targeted MS method is the SRM technique typically performed on a triple quadrupole instrument. However, the high analytical demands for performing clinical studies in combination with the extreme complexity of the samples involved are a serious challenge. The segmentation of the biomarker evaluation workflow has only partially alleviated these issues by differently balancing the analytical requirements and throughput at different stages of the process. The recent introduction of targeted high-resolution and accurate-mass analyses on fast sequencing mass spectrometers operated in parallel reaction monitoring (PRM) mode offers new avenues to conduct clinical studies and thus overcome some of the limitations of the triple quadrupole instrument. This article discusses the attributes and specificities of the PRM technique, in terms of experimental design, execution, and data analysis, and the implications for biomarker evaluation. The benefits of PRM on data quality and the impact on the consistency of results are highlighted and the definitive progress on the overall output of clinical studies, including high throughput, is discussed.
High resolution/accurate mass hybrid mass spectrometers have considerably advanced shotgun proteo... more High resolution/accurate mass hybrid mass spectrometers have considerably advanced shotgun proteomics and the recent introduction of fast sequencing capabilities has expanded its use for targeted approaches. More specifically, the quadrupole-orbitrap instrument has a unique configuration and its new features enable a wide range of experiments. An overview of the analytical capabilities of this instrument is presented, with a focus on its application to quantitative analyses. The high resolution, the trapping capability and the versatility of the instrument have allowed quantitative proteomic workflows to be redefined and new data acquisition schemes to be developed. The initial proteomic applications have shown an improvement of the analytical performance. However, as quantification relies on ion trapping, instead of ion beam, further refinement of the technique can be expected.
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