A robotic protocol for high-throughput processing of samples for selected reaction monitoring assays

Min Zhu; Pingbo Zhang; Minghui Geng-Spyropoulos; Ruin Moaddel; Richard D. Semba; Luigi Ferrucci

doi:10.1002/pmic.201600339

A robotic protocol for high-throughput processing of samples for selected reaction monitoring assays

Min Zhu, Pingbo Zhang, Minghui Geng-Spyropoulos, Ruin Moaddel, Richard D. Semba, Luigi Ferrucci

School of Medicine

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Selected reaction monitoring mass spectrometry (SRM-MS) is a sensitive and accurate method for the quantification of targeted proteins in biological specimens. However, the sample throughput and reliability of this technique is limited by the complexity of sample preparation, as well as instrumentation and data processing. Modern robotic equipment allows for rapid and accurate processing of large number of samples and makes SRM-MS assay applicable in epidemiological studies. Herein, we describe an automated sample processing platform developed in the context of an SRM-MS protocol for the assay of complement factor H protein and its variants in human plasma. We report detailed performance data on plasma digestion, sample cleanup and optimized robotic handling implemented on a Biomek^® NX^p Workstation. Method validation was assessed with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from 2.7 to 17.5% with median CV of 5.3%) and inter-day assay (CVs from 4.8 to 17.6 with median CV of 7.2%) for all peptides.

Original language	English (US)
Article number	1600339
Journal	Proteomics
Volume	17
Issue number	6
DOIs	https://doi.org/10.1002/pmic.201600339
State	Published - Mar 1 2017

Keywords

Absolute quantification
Automated sample preparation
Heavy isotope-labeled peptides
Reproducibility
SRM
Technology

ASJC Scopus subject areas

Biochemistry
Molecular Biology

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10.1002/pmic.201600339

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title = "A robotic protocol for high-throughput processing of samples for selected reaction monitoring assays",

abstract = "Selected reaction monitoring mass spectrometry (SRM-MS) is a sensitive and accurate method for the quantification of targeted proteins in biological specimens. However, the sample throughput and reliability of this technique is limited by the complexity of sample preparation, as well as instrumentation and data processing. Modern robotic equipment allows for rapid and accurate processing of large number of samples and makes SRM-MS assay applicable in epidemiological studies. Herein, we describe an automated sample processing platform developed in the context of an SRM-MS protocol for the assay of complement factor H protein and its variants in human plasma. We report detailed performance data on plasma digestion, sample cleanup and optimized robotic handling implemented on a Biomek{\textregistered} NXp Workstation. Method validation was assessed with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from 2.7 to 17.5% with median CV of 5.3%) and inter-day assay (CVs from 4.8 to 17.6 with median CV of 7.2%) for all peptides.",

keywords = "Absolute quantification, Automated sample preparation, Heavy isotope-labeled peptides, Reproducibility, SRM, Technology",

author = "Min Zhu and Pingbo Zhang and Minghui Geng-Spyropoulos and Ruin Moaddel and Semba, {Richard D.} and Luigi Ferrucci",

note = "Funding Information: This work was supported by the Intramural Research Program of the NIH on Aging, and NIH grants RO1 EY024596, RO1 AG027012 and R56 AG052973, the joint King Khaled Eye Specialist Hospital and Wilmer Eye Institute Research Grant Program. Publisher Copyright: {\textcopyright} 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim",

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AU - Semba, Richard D.

AU - Ferrucci, Luigi

N1 - Funding Information: This work was supported by the Intramural Research Program of the NIH on Aging, and NIH grants RO1 EY024596, RO1 AG027012 and R56 AG052973, the joint King Khaled Eye Specialist Hospital and Wilmer Eye Institute Research Grant Program. Publisher Copyright: © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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N2 - Selected reaction monitoring mass spectrometry (SRM-MS) is a sensitive and accurate method for the quantification of targeted proteins in biological specimens. However, the sample throughput and reliability of this technique is limited by the complexity of sample preparation, as well as instrumentation and data processing. Modern robotic equipment allows for rapid and accurate processing of large number of samples and makes SRM-MS assay applicable in epidemiological studies. Herein, we describe an automated sample processing platform developed in the context of an SRM-MS protocol for the assay of complement factor H protein and its variants in human plasma. We report detailed performance data on plasma digestion, sample cleanup and optimized robotic handling implemented on a Biomek® NXp Workstation. Method validation was assessed with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from 2.7 to 17.5% with median CV of 5.3%) and inter-day assay (CVs from 4.8 to 17.6 with median CV of 7.2%) for all peptides.

AB - Selected reaction monitoring mass spectrometry (SRM-MS) is a sensitive and accurate method for the quantification of targeted proteins in biological specimens. However, the sample throughput and reliability of this technique is limited by the complexity of sample preparation, as well as instrumentation and data processing. Modern robotic equipment allows for rapid and accurate processing of large number of samples and makes SRM-MS assay applicable in epidemiological studies. Herein, we describe an automated sample processing platform developed in the context of an SRM-MS protocol for the assay of complement factor H protein and its variants in human plasma. We report detailed performance data on plasma digestion, sample cleanup and optimized robotic handling implemented on a Biomek® NXp Workstation. Method validation was assessed with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from 2.7 to 17.5% with median CV of 5.3%) and inter-day assay (CVs from 4.8 to 17.6 with median CV of 7.2%) for all peptides.

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A robotic protocol for high-throughput processing of samples for selected reaction monitoring assays

Abstract

Keywords

ASJC Scopus subject areas

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