Quantitative assessment of multi-laboratory reproducibility of SDS-PAGE assays: Digestion pattern of beta-casein and beta-lactoglobulin under simulated conditions.
A digestion protocol was applied in triplicate by ten laboratories, simulating in vivo gastric and duodenal conditions. The intra- and inter-laboratory variability in the kinetics of protein degradation was quantified, focussing on the digestion of beta-casein under gastric conditions, and of beta-lactoglobulin (beta-Lg) under duodenal conditions. The addition of surfactants such as phosphatidylcholine (PC) in the digestion mix was also evaluated. Identification and quantification of peptide bands on SDS-PAGE gels formed the basis for analysis. An average intensity loss of 69% (SD=13.5) at 5 min (89% at 10 min, with SD=5.5) was observed for beta-casein, whereas the beta-Lg duodenal digestion showed an 82% loss at 30 min (SD=14.2). Constant rates of first-order reactions showed that for fast reactions, inaccuracies in the time of first sampling contributed to the variability, which were also affected by image quality, saturation, and the splitting of time courses across gels. Breakdown products for beta-casein included ten other polypeptides, with four detected in all and two in most gels, and for beta-Lg ten polypeptides, with five detected in most, and two in two-third of the cases. Addition of PC in the gastric phase led to beta-Lg intensity loss only a quarter as large as without PC and altered beta-Lg proteolysis in the duodenal compartment.
Defernez
Marianne
M
Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK. marianne.defernez@bbsrc.ac.uk
Mandalari
Giuseppina
G
Mills
Elizabeth Noami Clare
EN
D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004063:Digestion / Q000502:physiology*; D004386:Duodenum / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D005753:Gastric Mucosa / Q000378:metabolism; D007700:Kinetics; D007753:Laboratories / Q000592:standards*; D007782:Lactoglobulins / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D007782:Lactoglobulins; D012967:Sodium Dodecyl Sulfate; D002918:Chymotrypsin; D014357:Trypsin