Dataset Information

Comparison of targeted mass spectrometry techniques with an immunoassay: a case study for HSP90 alpha, part 1

ABSTRACT: We developed 2D-LC-MS/MS-based SRM and PRM assays to measure HSP90 alpha in serum and compared the results to a commercially available immunoassay (ELISA). Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90 alpha in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation. To rule out that the observed differences in SRM and PRM are due to different mass spectrometry systems, the SCX fractions were measured on the same high-resolution instrument (Orbitrap) in the ion trap mode (IT-PRM); such measurements showed that intense co-eluting signals were present in the SRM method, but these interfering peaks were eliminated in the high-resolution PRM mode. Thus, this report shows that it is possible to measure ng/mL levels of HSP90 alpha in a reproducible, selective and sensitive way using PRM. This opens up the possibility to quantify low levels of multiple proteins in complex samples based on a fractionation strategy on tryptic peptides followed by SRM and PRM.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Coskun Guzel

LAB HEAD: Theo Luider

PROVIDER: PXD006618 | Pride | 2018-07-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	1fmol_HSP90_SILpepmix_PRM (F130247).mzid_1fmol_HSP90_SILpepmix_PRM_(F130247).pride.mgf.gz	Mzid
	1fmol_HSP90_SILpepmix_PRM (F130247).pride.mztab.gz	Mztab
	1fmol_HSP90_SILpepmix_PRM.raw	Raw
	1fmol_HSP90_SILpepmix_PRMF130247.mzid.gz	Mzid
	1fmol_HSP90_SILpepmix_PRMF130247.mzid_1fmol_HSP90_SILpepmix_PRM_F130247.MGF	Mzid

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Publications

Comparison of Targeted Mass Spectrometry Techniques with an Immunoassay: A Case Study for HSP90α.

Güzel Coşkun C Govorukhina Natalia I NI Stingl Christoph C Dekker Lennard J M LJM Boichenko Alexander A van der Zee Ate G J AGJ Bischoff Rainer P H RPH Luider Theo M TM

Proteomics. Clinical applications 20171030 1

<h4>Purpose</h4>The objective of this study is to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay.<h4>Experimental design</h4>A 2D-LC-MS/MS-based SRM and PRM assay is developed for quantitative measurements of HSP90α in serum. Forty-three control sera are compared by SRM, PRM, and ELISA following the manufacturer's instructions. Serum samples are trypsin-digested and fractionated by strong cation exchange chromatography prior to SRM and ...[more]

PMID: 28975736

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Project description:Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validation and clinical translation of protein biomarkers discovered by global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of specific endogenous (Endo) peptides with those of stable isotope labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrants sensitive and precise measurements required for targeted MS assays. Compared to MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensate for different ion suppression among transitions of a target peptide. However, PRM still suffers different ion suppressions between Endo and SIL peptides due to poor spray stability as the Endo and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed as wideband PRM (WBPRM), designed for high-throughput targeted MS approach. WBPRM employs a wide isolation window for simultaneous fragmentations of both Endo and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of target peptides. Compared to PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and accuracy of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a short experiment time. Its adaptability to a MS method provided by the manufacture makes it a facile method to implement for MS based assays, particularly in complex biological samples, where the needs for higher accuracy, sensitivity and efficiency are paramount.