Quantitative protein mass-spectrometry requires a standardized pre-analytical phase.

OBJECTIVES: Quantitative protein mass-spectrometry (QPMS) in blood depends on tryptic digestion of proteins and subsequent measurement of representing peptides. Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery is unknown. In our laboratory serum samples are the preferred matrix for QPMS measurement of multiple apolipoproteins. In this study, we investigated the effect of different matrices on apolipoprotein quantification by mass spectrometry. METHODS: Blood samples were collected from 44 healthy donors in Beckton Dickinson blood tubes simultaneously for serum (with/without gel) and plasma (heparin, citrate or EDTA). Nine apolipoproteins were quantified according to standard operating procedure using value-assigned native serum calibrators for quantitation. Tryptic digestion kinetics were investigated in the different matrices by following formation of peptides for each apolipoprotein in time, up to 22 h. RESULTS: In citrate plasma recovery of apolipoproteins showed an overall reduction with a bias of -14.6%. For heparin plasma only -0.3% bias was found compared to serum, whereas for EDTA-plasma reduction was more pronounced (-5.3% bias) and variable with >14% reduction for peptides of apoA-I, A-II and C-III. Digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma. CONCLUSIONS: Plasma anticoagulants affect QPMS test results. Heparin plasma showed comparable results to serum. Reduced concentrations in citrate plasma can be explained by dilution, whereas reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency. The results highlight the importance of a standardized pre-analytical phase for accurate QPMS applications in clinical chemistry.

Homeostasis and function of goblet cells during rotavirus infection in mice.

Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.

Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice.

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.