Clinical Chemistry
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Clinical Chemistry 52: 73-81, 2006. First published October 27, 2005; 10.1373/clinchem.2005.056861
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2006;52:73-81.)
© 2006 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Application of Proteomic Technology in Identifying Pancreatic Secretory Trypsin Inhibitor Variants in Urine of Patients with Pancreatitis

Leena Valmu1,a, Annukka Paju1, Marko Lempinen2, Esko Kemppainen3 and Ulf-Håkan Stenman1

1 Department of Clinical Medicine, Division of Clinical Chemistry, Biomedicum, University of Helsinki, Helsinki, Finland.
2 Department of Surgery, Clinic of Transplantation and Liver Surgery, and3 Second Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland.

aAddress correspondence to this author at: Department of Clinical Medicine, Division of Clinical Chemistry, Biomedicum Helsinki, University of Helsinki, PO Box 63 (Haartmaninkatu 8), Helsinki FIN-00014, Finland. Fax 358-9-47171731; e-mail leena.valmu{at}helsinki.fi.

Background: Although the analysis of genetic variability has traditionally been performed with molecular genetic techniques, the development of proteomic technology has raised the possibility of analyzing genetic variants at the protein level. This method provides additional information about posttranslational modifications and differences in expression. We used mass spectrometry to characterize 3 variants of the peptide encoded by the serine protease inhibitor Kazal type 1 (SPINK1) gene, pancreatic secretory trypsin inhibitor (PSTI). A genetic variant of PSTI, N34S, is associated with the development of pancreatitis.

Methods: We used a quadrupole/time-of-flight hybrid mass spectrometer equipped with an electrospray ionization source to analyze the molecular identity of PSTI purified from the urine of 12 patients with pancreatitis and from 3 controls. We also developed a rapid small-scale capture procedure to isolate and analyze PSTI from small volumes of urine.

Results: The mutations responsible for mass shifts of different PSTI variants could be verified. We observed differences in the expression of different variants as well as a novel proteolytic fragment of PSTI. Small-scale magnetic bead–mediated immunoaffinity chromatography PSTI enabled easy and rapid purification from small urine volumes, facilitating mass spectrometric analysis with adequate sensitivity.

Conclusions: Pancreatitis-related PSTI variants occurring at nanomolar concentrations in urine can be detected and quantified by immunoaffinity purification and mass spectrometry. In addition, the N34S variant occurs at higher concentrations than the wild type. This finding casts new light on the possible role of PSTI as a cause of hereditary pancreatitis.






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