Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

Abstract

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno-capture of ProGRP in 96-wells microtiter plates coated with the mAb E146. After immuno-capture and thorough rinse, trypsin was added for in-well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC-MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9-17.5%), accuracy (3.0-6.6%), and linearity (r(2) = 0.995) in the tested range (0.5-50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC-MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno-capture LC-MS method was superior with respect to sensitivity. This illustrates the large potential of immuno-capture sample preparation prior to LC-MS in protein biomarker quantification.