Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 310

this protocol, analysis is exemplified using a Q-TOF instrument
equipped with a nano-electrospray ion source. Similarly, other
separation techniques, in addition to LC, may also be suitable,
with capillary electrophoresis showing impressive results ( 13 ) .
Figure 19.1 illustrates the complexity of the urinary peptidome
as analysed with this LC-MS workflow; hundreds (or even thou-
sands) of peptides can be detected in a 30 min gradient LC-MS
run from the analysis of 50 l of urine (by extrapolation). Hun-

dreds of MS/MS spectra can be collected and thousands of
these small peptides may be present in urine ( 9 , 11 , 14 ) , making
the peptidome of this fluid an attractive, yet largely unexplored,
source of potential biomarkers of disease. With minor modifica-
tions the utility of this protocol could also be extended to analyse
larger polypeptides and proteins.

μ

2. Materials

2.1.Reversed-Phase
Solid-Phase
Extraction

1. Peptide mixture obtained from the digestion of yeast eno-
lase.
2. Reversed-phase (RP) solid-phase extraction (SPE) cartridges
(HLB Oasis 94226, Waters, Milford, MA).
3. SPE vacuum manifold (optional).
4. RP conditioning solution: acetonitrile (ACN) ( see Note 1 ).
5. RP equilibration solution: 0.1% (v/v) TFA/5% (v/v) ACN.
6. RP elution solution: 0.1% (v/v) TFA/60% (v/v) ACN.

2.2.StrongCation
ExchangeExtraction

1. Strong cation exchange (SCX) magnetic beads (Dyna beads,
Invitrogen).
2. Magnetic rack separator for 1.5 ml tubes (e.g. Invitrogen cat
no. CS1500) and for 96-well plates.
3. SCX conditioning solution: 1 M NaCl/50 mM ammonium
bicarbonate, pH 8.8 (ammonium solutions are volatile and
should be prepared fresh).
4. SCX loading solution: 0.1% (v/v) TFA/20 (v/v)%
ACN/0.01% Tween-20.
5. SCX wash solution: 0.1% (v/v) TFA/20 (v/v)% ACN.
6. SCX elution solution: 500 mM ammonium acetate in 20%
(v/v) ACN.

1. Solvent A: 0.1% formic acid in LC-MS grade water.
2. Solvent B: 0.1% formic acid in LC-MS grade ACN.
3. Trap column (Symmetry C18, 180

2.3.LC-MS/MS(see
Note2)

μ m × 20 mm, Waters)

( see Note 2 ).