25. Reagents dissolved in anhydrous acetonitrile are stable for
1 week when stored at À20
C.
26. To calculate the total volume of the combined sample, multiply
the number of samples from the beginning (in this case 10) by
the volume of protein dissolved in TEAB, the volume of added
TCEP, the volume of added IAA, the volume of added trypsin,
the volume of added TMT mass tag, the volume of added
hydroxylamine, and add all together. If the total volume of
the combined sample is, e.g., 1500 μL and at the beginning
there were 10 samples each of 100 μg of protein, you should
divide 1500 by 10 and put 150 μL to each new tube. This way
you will have peptide aliquots with 100 μg of protein digest.
27. The aliquots may be frozen at À80
C until the next step or
you can proceed with peptide fractionation.
28. Measuring protein digest concentration by quantitative colorimetric peptide assay will give us information if we have at least
100 μg of TMT-labeled peptides in the aliquot. This amount is
optimal for peptide fractionation and next for LC–MS/MS
analysis.
29. Do not exceed recommended centrifugation speeds.
30. Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery during all steps of fractionation
procedure.
31. The aliquots may be frozen at À80
C until the next step or
you can proceed with LC–MS/MS analysis.
32. The LC–MS/MS system used here was Proxeon EASY-nLC II
liquid chromatography system (Thermo Fisher Scientific) coupled to LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher
Scientific). Remember to clean, calibrate, and tune MS instrument regularly and use fresh LC buffers to improve the performance of the system.
33. These organisms were selected according to phylogenetic
similarity.
34. The type of analysis performed on your result data depends on
the aim of the experiment, i.e., comparison of two development stages or comparison between control and treatment
samples.
Acknowledgments
This work was supported by the “Development Program of the
University of Warmia and Mazury in Olsztyn,” cofinanced by the
European Union under the European Social Fund from the Operational Program Knowledge Education Development. R. S. is a
recipient of a scholarship from the program Interdisciplinary
74
Robert Stryin ´ ski et al.
1 week when stored at À20
C.
26. To calculate the total volume of the combined sample, multiply
the number of samples from the beginning (in this case 10) by
the volume of protein dissolved in TEAB, the volume of added
TCEP, the volume of added IAA, the volume of added trypsin,
the volume of added TMT mass tag, the volume of added
hydroxylamine, and add all together. If the total volume of
the combined sample is, e.g., 1500 μL and at the beginning
there were 10 samples each of 100 μg of protein, you should
divide 1500 by 10 and put 150 μL to each new tube. This way
you will have peptide aliquots with 100 μg of protein digest.
27. The aliquots may be frozen at À80
C until the next step or
you can proceed with peptide fractionation.
28. Measuring protein digest concentration by quantitative colorimetric peptide assay will give us information if we have at least
100 μg of TMT-labeled peptides in the aliquot. This amount is
optimal for peptide fractionation and next for LC–MS/MS
analysis.
29. Do not exceed recommended centrifugation speeds.
30. Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery during all steps of fractionation
procedure.
31. The aliquots may be frozen at À80
C until the next step or
you can proceed with LC–MS/MS analysis.
32. The LC–MS/MS system used here was Proxeon EASY-nLC II
liquid chromatography system (Thermo Fisher Scientific) coupled to LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher
Scientific). Remember to clean, calibrate, and tune MS instrument regularly and use fresh LC buffers to improve the performance of the system.
33. These organisms were selected according to phylogenetic
similarity.
34. The type of analysis performed on your result data depends on
the aim of the experiment, i.e., comparison of two development stages or comparison between control and treatment
samples.
Acknowledgments
This work was supported by the “Development Program of the
University of Warmia and Mazury in Olsztyn,” cofinanced by the
European Union under the European Social Fund from the Operational Program Knowledge Education Development. R. S. is a
recipient of a scholarship from the program Interdisciplinary
74
Robert Stryin ´ ski et al.