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All figures (29)
Figure 24. Fold change (log2) of immune proteins, growth factors and MEP1A in the rejection group, AR1 (●) AR2 (×) AR3 (♦) AR4 (+) AR5 (■) AR6 (▲), from baseline to Biopsy Proven Acute Rejection (BPAR). The center point (Clinically stable) is 7-11 days before BPAR, at stable serum creatinine levels.
Table 4. Different platforms used for peptide/protein identification
Figure 7. Illustration of 18O-incorporation by two different mechanisms, amide bond cleavage and carboxyl oxygen exchange (From reference [68] with permission).
Figure 8. Schematic workflow for peptide/protein identification after LC-MS/MS
Table 2. Overview of key variables and variability in different steps of the workflow
Figure 23. Scheme of the samples analyzed and compared from each patient
Figure 5. A model of the orbitrap mass analyzer (from reference [52] with permission)
Figure 19. Overview of the integrated digestion and labeling procedure on immobilized trypsin beads. The sample is first digested in ammonium hydrogen carbonate buffer (in H216O) followed by evaporation. The sample is then reconstituted in H218O containing buffer (pH 6) and extra trypsin beads (separate vial). Labeling is then carried out in the same vial as the digestion, and is stopped by removing trypsin beads and adding 8 M urea. Corresponding 16O- and 18O-labeled samples are mixed in a 1:1 ratio before LC-MS/MS analysis
Figure 25. Boxplot showing fold change (log2) of immune proteins, growth factors and MEP1A from baseline to AR in the AR-group compared with the control group
Figure 9. Schematic outline of the main steps of the method workflow and solvents used in the respective steps.
Figure 15. Different digestion reactors (A-D) which were investigated
Figure 6. Schematic outline of the main components of the LTQ Orbitrap (from reference [52] with permission)
Figure 12. The upper chromatogram shows gradient elution separation of a tryptic digest of cyt c on a HILIC column starting at 80 % MeCN. The lower chromatogram shows separation with 95 % MeCN as gradient starting conditions. Both samples were dissolved in its respective starting mobile phase and equal concentrations of cyt c were used.
Figure 16. Scheme of the on-line coupled instrumentation system
Figure 3. Structure of the ZIC-HILIC stationary phase
Figure 11. Gel electrophoresis of 2 urine samples depleted for HSA (red marking). Gel A was pure urine, gel B was spiked with HSA. Lane 1 in the gels shows crude urine prior to depletion. Lane 2-6 shows the flow-through fractions with depleted urine and lane 7 is a washing step. Lane 8 shows the fractions where trapped HSA from the samples is eluted.
Figure 18. Average 18O/16O-ratios (±SD) of 12 BSA / cyt c peptides using labeling buffer of pH 5, 6, 7, 8 or 9 (n = 3). Reaction time was 2 hours for all samples
Figure 1. Overview of cells and mediators involved in acute rejection (from reference [6] with permission).
Figure 21. Orbitrap mass spectra of six 18O-labeled BSA peptides before mixing with unlabeled peptides. (a) LVTDLTK, (b) AEFVEVTK, (c) YLYEIAR, (d) HLVDEPQNLIK, (e) KVPQVSTPTLVEVSR, (f) LGEYGFQNALIVR. Spectra were obtained from urine spiked with BSA, digested and labeled by immobilized trypsin
Figure 13. Chromatograms of corresponding fractions analyzed in the second dimension from two similar experiments distinguished by the use of nano and micro columns respectively. The samples contained comparable amounts of total protein.
Table 5. Intensity change of tryptic peptides from BSA and cyt c after replacing tryptic digestion in-solution with digestion using immobilized trypsin beads
Figure 22. Ratio distribution of all 18O/16O-labeled peptides identified (x-axis) in urine from a kidney transplanted patient (n = 2). Both tryptic digestion and 18O/16O-labeling were done using immobilized trypsin
Figure 10. Protein recovery from centrifugal filters (5 kDa cut-off) using different volumes of water (×), 25 mM TrisHCl (▲), 25 mM TrisHCl + wash (∆), 8 M Urea (♦), 10 mM TrisHCl/150 mM NaCl (■), 10 mM TrisHCl/150 mM NaCl + wash (□) to redissolve the proteins after centrifugation
Figure 17. Average 18O/16O-ratios (±SD) of 12 BSA / cyt c peptides at different time points (n = 3). Labeling was done at pH 6
Table 3. Identified peptides of BSA in urine and buffer
Figure 14. Plot of retention time (tR) of peaks in the 2nd dimension (RP) vs. fraction number from the 1st dimension (HILIC). The sample used was from a kidney transplanted patient
Table 1. Protein recoveries from urine using various sample preparation methodsa
Figure 4. The most common cleavage sites following CID fragmentation. (This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license)
Figure 20. Average 18O/16O-ratios (±SD) for 12 BSA/cyt c peptides (n = 3). (a) Tryptic digestion in-solution/labeling on immobilized trypsin. (b) Both tryptic digestion and labeling on immobilized trypsin beads. (c) Both tryptic digestion and labeling on immobilized trypsin beads, extra beads added between the steps
Journal Article
•
DOI
•
2-D hydrophilic interaction liquid chromatography-RP separation in urinary proteomics--minimizing variability through improved downstream workflow compatibility.
[...]
Håvard Loftheim
1
,
Thien Duc Nguyen
1
,
Helle Malerod
1
,
Elsa Lundanes
1
,
Anders Åsberg
1
,
Léon Reubsaet
1
- Show less
+2 more
•
Institutions (1)
University of Oslo
1
01 Mar 2010
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Journal of Separation Science