The development of SRM assays is transforming proteomics research

Manes, Nathan P.; Nita‐Lazar, Aleksandra

doi:10.1002/pmic.201600366

Cited by 4 publications

(17 citation statements)

References 7 publications

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“…All protein sequences were retrieved from UniProt 12 . The target proteins are tabulated in the file “Targeted Proteins.xlsx” available at Panorama Public 13 .…”

Section: Methodsmentioning

confidence: 99%

“…In total, 851 crude light ( i.e ., not labeled with stable isotopes) peptides were purchased for qualitative analyses, and 279 purified quantitated heavy-labeled ( 13 C 6 15 N 4 Arg, 13 C 6 15 N 2 Lys) internal peptide standards were purchased for quantitative analyses. The target peptides are tabulated in the file “Targeted Peptides.xlsx” available at Panorama Public 13 . Of the 279 quantitated peptides, 29 are phosphopeptides for phosphoprotein quantitation.…”

Section: Methodsmentioning

confidence: 99%

“…The BMDM-derived samples were analyzed in technical duplicate ( = 16 × 14 runs); the other samples (“B0-1”, “B0-2”, and “B0-3”) were each analyzed once ( = 3 × 14 runs). The resulting data were uploaded to Panorama Public 30 (ProteomeXchange 31 ID: PXD031697; 10.6069/44s8-9f68) (Table 2 ) 13 . LC-PRM was also performed using the heavy-labeled peptide standards alone to develop scheduled LC-PRM assays.…”

Section: Methodsmentioning

confidence: 99%

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Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

et al. 2022

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The Toll-like receptor (TLR) and chemotaxis pathways are key components of the innate immune system. Subtle variation in the concentration, timing, and molecular structure of the ligands are known to affect downstream signaling and the resulting immune response. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but such simulations require protein concentration values as model parameters. Here we report the development and application of targeted mass spectrometry assays to measure the absolute abundance of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. Two peptides per protein were quantified, if possible. The protein abundance values ranged from 1,332 to 227,000,000 copies per cell. They moderately correlated with transcript abundance values from a previously published mouse macrophage RNA-seq dataset, and these two datasets were combined to make proteome-wide abundance estimates. The datasets produced during this investigation can be used for pathway modeling and simulation, as well as for other studies of the TLR and chemotaxis pathways.

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“…All protein sequences were retrieved from UniProt 12 . The target proteins are tabulated in the file “Targeted Proteins.xlsx” available at Panorama Public 13 .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

et al. 2022

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“…An MRM assay to measure 12 different isoforms of GTPase simultaneously in the presence of agonists or inhibitors was developed and utilized . Thus, there are a significant number of studies available now, which have been reviewed in other articles, that provide us with a complete picture of current applications of targeted proteomics in different areas of biology . These successful attempts provide a proof of principle to take a leap into what is required to be studied regardless of the availability of the reagents.…”

Section: Targeted Proteomics: What Does It Bring For Researchers At Bmentioning

confidence: 99%

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

Arora

Somasundaram

2019

BioEssays

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While mass spectrometry (MS)‐based quantification of small molecules has been successfully used for decades, targeted MS has only recently been used by the proteomics community to investigate clinical questions such as biomarker verification and validation. Targeted MS holds the promise of a paradigm shift in the quantitative determination of proteins. Nevertheless, targeted quantitative proteomics requires improvisation in making sample processing, instruments, and data analysis more accessible. In the backdrop of the genomic era reaching its zenith, certain questions arise: is the proteomic era about to come? If we are at the beginning of a new future for protein quantification, are we prepared to incorporate targeted proteomics at the benchside for basic research and at the bedside for the good of patients? Here, an overview of the knowledge required to perform targeted proteomics as well as its applications is provided. A special emphasis is placed on upcoming areas such as peptidomics, proteoform research, and mass spectrometry imaging, where the utilization of targeted proteomics is expected to bring forth new avenues. The limitations associated with the acceptance of this technique for mainstream usage are also highlighted. Also see the video abstract here https://youtu.be/mieB47B8gZw.

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“…The adaptation of targeted data acquisition in the form of selected reaction monitoring (SRM), approximately a decade ago, was initially motivated by the requirement for robust and sensitive quantification of proteins 50 . Numerous LC-MS workflows employ shotgun LC-MS; however, many others require a significantly higher reproducibility, sensitivity, accuracy, and precision of SRM 51 . SRM, also known as multiple reaction monitoring, uses triple Quadrupole (QD) (Figure 5 ), where molecular ions are selected in Q1, collision-activated dissociation fragmentation is performed in Q2, and unique fragments ions are evaluated in Q3 52 .…”

Section: Principles and Instrumentationmentioning

confidence: 99%

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

Huang¹,

Chen²,

He³

et al. 2017

Theranostics

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There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed.

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The development of SRM assays is transforming proteomics research

Cited by 4 publications

References 7 publications

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

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