Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 312

4. Nanoflow analytical RP column (BEH130 C18, 100 μ m ×

100 mm, Waters).
5. LC-MS/MS system (this protocol will be exemplified with
a nanoAcquity UPLC connected online with a Q-TOF Pre-
mier, Waters).

3. Methods

3.1.UrineSample
Preparation(see
Note3)

1. As soon as possible after collection, urine samples should be
refrigerated to 0 ◦ C by placing them on ice.
2. Centrifuge specimens at 10,000 g for 10 min in a centrifuge

refrigerated to 4 ◦ C. This step removes cells shedded by the
urogenital track and other debris.
3. Specimens should be aliquoted into 1 ml aliquots and stored
at

×

80 ◦ C until the time of analysis (optional).
4. On the day of analysis urine specimens should be defrosted
at room temperature. Defrosted specimens should be stored
at 0 ◦ -4 ◦ C until extraction is performed.
5. Add a known amount of peptide mixture to the samples.
These will serve as internal standards for quantification. For
human urine, 1 pmol of peptide derived from yeast enolase
spiked into 1 ml urine can serve as a convenient internal
standard peptide mixture.
6. Acidify specimens by adding 10

−

μ l 10% TFA to 1 ml urine.

7. Centrifuge at 12,000 × g for 10 min ( see Note 4 ).

3.2.Extraction
ofPeptidesbyRP
SPE(see Note5)

1. Place RP SPE cartridges in a vacuum manifold ( see Note 6 ).
2. Fill cartridges with RP conditioning solution. Apply vacuum
to the manifold so that the flow rate of mobile phase through
the cartridge is about 2 ml/min. The steps for SPE extrac-
tion are conditioning of the cartridge, equilibration, sample
loading, washing and elution. In each case the solvent from
the previous step should have completely passed through
the column before adding the solvent of the next step ( see
Note 7 ).
3. Conditioning: apply a total of 10 ml of RP conditioning
solution to the cartridge.
4. Equilibration: add 10 ml of RP equilibration solution.
5. Sample loading: add acidified urine specimens to the equili-
brated SPE RP cartridges.
6. Washing: when specimens have passed through cartridges,
wash the cartridge by adding 10 ml of RP equilibration