An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000 mu g/L. The limit of quantitation (LOQ) was 0.5 mu g/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.Peer reviewe

Comparative Metaproteomics and Diversity Analysis of Human Intestinal Microbiota Testifies for Its Temporal Stability and Expression of Core Functions

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