Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 316

5. Normalise these values to those of internal standard peptides
( see Note 15 ).
4. Notes
1. All reagents should be prepared using LC-MS or HPLC
grade solvents.
2. LC-MS/MS analysis is exemplified here using a Waters
Q-TOF MS/MS instrument connected online with a
nanoflow UPLC system (nanoAcquity). Any other instru-
mentation capable of performing MS/MS experiments and
of LC separations at nanolitre per minute flow rates should
also be adequate. Similarly, other C18 trap and analytical
columns capable of accepting nanoflow per minute flow
rates should also be adequate.
3. These sample preparation steps are designed to analyse
small peptides. For the analysis of larger polypeptides and
proteins, it is necessary to introduce a proteolytic step (e.g.
using sequencing grade trypsin) prior to further sample
preparation, so as to generate protein-derived peptides of a
size compatible with the rest of the protocol. This optional
proteolytic step may be performed between Steps 5 and 6
in Section 3.1 .
4. This second centrifugation step may be omitted when sam-
ples are processed fresh without a freezing cycle.
5. The RP SPE step separates organic hydrophobic molecules,
which includes peptides and proteins, from inorganic salts.
This desalting step makes peptides ready for further extrac-
tion by SCX.
6. Manual application of mobile phases through SPE car-
tridges using syringes is an alternative to a vacuum man-
ifold. However, by using a vacuum manifold the procedure
is more efficient and several samples can be processed in
parallel.
7. Cartridges should not be allowed to dry. Doing so may
result in low recovery of peptides.
8. This SCX step separates peptides from hydrophobic organic
acids, which dominate MS spectra of unprocessed urine and
mask the presence of small peptides in urine.
9. This protocol uses magnetic SCX beads because of conve-
nience of use. Other SCX material (i.e. silica based SCX
beads) has been used in this laboratory with good results.
10. Tween-20 is a detergent that helps avoiding bead aggrega-
tion that may occur as a result of hydrophobic interactions