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

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 PRM.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Coskun Guzel  

LAB HEAD: Theo Luider

PROVIDER: PXD006569 | Pride | 2022-03-10

REPOSITORIES: Pride