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

Abstract

Purpose: The objective of this study is to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. Experimental design: 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 PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability, and reproducibility of the SRM, PRM, and ELISA are determined. Results: PRM data obtained by high-resolution MS correlate better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, and ELISA) are able to quantify HSP90α in serum at the ng mL -1 level, the use of PRM on a high-resolution mass spectrometer reduces variation and shows comparable sensitivity to immunoassay. Conclusions and clinical relevance: Using fractionation, it is possible to measure ng mL -1 levels of HSP90α in a reproducible, selective, and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders.

Quantification of Calcyclin and Heat Shock Protein 90 in Sera from Women with and without Preeclampsia by Mass Spectrometry

“…For S100A6 , a relatively low molecular weight protein (≈10 kDa) only the single signature peptide LQDAEIAR was used because other tryptic S100A6 peptides contain amino acids that are prone to oxidation (methionine, cysteine), acetylation, phosphorylation or are too long, that is, more than 20 amino‐acids. The single signature peptide YIDQEELNK was used for HSP90 measurements, because almost identical results were obtained for another HSP90 peptide DQVANSAFVER as explained in our previous paper . In this study, an agreement between ELISA and PRM results for HSP90 was shown.…”

“…All chemicals used for SCX fractionation were purchased from Sigma‐Aldrich (St Louis, MO). As shown previously, 50 μL fractions (180 fractions in total for each serum sample) were automatically collected in 384‐well plates (VWR, Amsterdam, the Netherlands) and sealed with an adhesive aluminum foil (VWR, Amsterdam, the Netherlands). Fractions were dried down in SpeedVac concentrator (RVT4104, Scientific Savant, San Jose, CA) and subsequently stored at −20 °C until further analysis.…”

“…We investigated whether these two proteins behave differently in patients with PE as compared to pregnant normotensive controls. A serum‐based 2D LC/MS PRM assay was used to quantify both proteins. In addition, we developed a double immunofluorescence staining with antibodies against S100A6 and HSP90 to show that both proteins S100A6 and HSP90 are present in trophoblast cells.…”

Neoantigens in Chronic Obstructive Pulmonary Disease and Lung Cancer: A Point of View

“…Mass spectrometry has, in addition to the potential to identify proteins in the presence or absence of databases, the inherently present possibility of quantification of proteins and peptides in a relatively sensitive way . This opens possibilities to detect missense mutations in antigens and even neo‐antigens in biopsies and body fluids.…”

“…Without sample preparation, most often microgram per mL biofluid can be reached. If affinity separations (e.g., by specific column chromatography materials, binders such as antibodies or affimers) are applied one can reach the ng mL −1 biofluid or pg protein per g tissue . These improved sample preparations and improved technology in quadrupole and high resolution detection in advanced mass spectrometers (hardware and software) will enable the large scale detection and quantitation of mutated proteins in the near future.…”