Application of proteomic technology in identifying pancreatic secretory trypsin inhibitor variants in urine of patients with pancreatitis.
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.
Valmu
Leena
L
Department of Clinical Medicine, Division of Clinical Chemistry, Biomedicum, University of Helsinki, Helsinki, Finland. leena.valmu@helsinki.fi
Paju
Annukka
A
Lempinen
Marko
M
Kemppainen
Esko
E
Stenman
Ulf-Håkan
UH
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D050500:Pancreatitis, Chronic / Q000235:genetics / Q000652:urine*; D040901:Proteomics; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014359:Trypsin Inhibitor, Kazal Pancreatic
D002352:Carrier Proteins; C486642:SPINK1 protein, human; D014359:Trypsin Inhibitor, Kazal Pancreatic