In this paper, we have used a newly developed immunocapture and LC-MS method to demonstrate for the first time the presence of protein isoforms 1 and 3 of the small cell lung cancer (SCLC) marker progastrin-releasing peptide (ProGRP) in sera. In addition, the method allows for indirect determination of the relative presence of the other known isoform of ProGRP, also known as ProGRP isoform 2. This new method is able to determine total ProGRP as a marker in sera at clinically relevant levels and to differentiate between isoforms at the low-pM level through combining selective sample preparation by immunoextraction, tryptic digestion, and separation followed by detection with LC-SRM-MS of the signature peptides, NLLGLIEAK (total ProGRP), LSAPGSQR (ProGRP isoform 1), and DLVDSLLQVLNVK (ProGRP isoform 3), with accuracies ≤ 25% for lower limit of quantification (LLOQ) and precisions ≤ 33%. By analyzing serum samples from four patients diagnosed with SCLC using the here described new and fully validated method, the ability is shown to both determine total ProGRP concentration and to differentiate between ProGRP isoforms 1 and 3 in one single run. Quantification of various ProGRP isoforms in one single run may be helpful for further understanding of the underlying biochemical processes in SCLC and differentiation of small cell lung cancer.
Progastrin-releasing peptide (proGRP) is a precursor of gastrin-releasing peptide, a hormone which is secreted from neuroendocrine cells. It has been shown to be a useful serum marker for small cell lung cancer. We raised monoclonal antibodies (MAbs) against proGRP with the primary aim of establishing a sensitive immunoassay. Immunization was performed with recombinant proGRP (amino acids 31–98) conjugated to thyroglobulin or with a DNP-modified peptide. Seven of the MAbs recognizing both recombinant and cell line-derived peptide were characterized and epitope-mapped. Based on cross-inhibition studies the antibodies could be categorized into three main groups. The molecular epitope assignment was studied by using phages displaying proGRP peptides, random peptide libraries displayed on phage and by pepscan analysis utilizing 10-mer biotinylated peptides. Two of the MAbs (E146, E172) bound to a defined region on the N-terminal part of proGRP(31–98), three recognized conformational-dependent epitopes in the middle of the peptide (E179, E180, E181) and two bound to the C-terminal part (E149, E168). Consensus sequences were obtained for MAbs E146, E149 and E168. The binding kinetics of the MAbs was determined by surface plasmon resonance, and a time-resolved immunofluorometric assay was established.
BACKGROUND: Small cell lung cancer accounts for approximately 20% of new cases of lung cancer, and advanced disease is prevalent at the time of diagnosis. Neuron-specific enolase (NSE) has been the primary tumor marker in small cell lung cancer but it has relatively low sensitivity in early-stage disease. Progastrinreleasing peptide (proGRP) is a promising alternative or complementary marker for NSE. We have previously described a time-resolved immunofluorometric assay (TR-IFMA) for proGRP that lacked the necessary sensitivity and robustness for use in the routine clinical laboratory. Herein we describe the development of an improved assay using a novel monoclonal antibody pair.
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.
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