Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 309

the urogenital track or as a result of incomplete reabsorption of
plasma proteins by kidney cells. Thus the urinary proteome con-
tains a mixture of plasma and kidney proteins, whose ratios may
be altered in disease ( 8 , 9 ) .
As with blood, urine contains proteases that actively digest
proteins into small peptides, which also appear in urine in large
quantities ( 10 ) (proteases are also present on luminal membranes
of renal epithelial cells and are therefore in contact with the 'pre-
urine'). These small peptides may not merely be waste products in
their route to excretion. Indeed, it has been postulated that, since
they are present in the tubular fluid (the 'pre-urine' in contact
with tubular kidney cells), some of these peptides may have phys-
iological roles by acting on luminal receptors of kidney tubular
cells ( 11 ) .
Protocols for urinary proteomics are in general designed
to analyse large polypeptides (>10 kDa) and these are either
based on size exclusion techniques (ultrafiltration, dialysis or
gel filtration) or on protein precipitation with organic solvents
or acids ( 12 ) . These sample preparation techniques are needed
because urine contains large amounts of organic and inorganic
salts that interfere with downstream protein analysis by 2D gel
electrophoresis or by direct LC-MS/MS. However, extraction
techniques based on size exclusion or precipitation, in addition
to removing salts, also remove small peptides, which are therefore
not analysed in most urinary proteomics studies.
The present protocol is designed for enriching urinary pep-
tides in a form that is compatible with downstream analysis by
LC-MS and LC-MS/MS with electrospray as the ion source.
These peptides have the same mass to charge ratio ( m / z )and
hydrophobicity as organic acids, which are present in large quan-
tities in urine, and whose signals, therefore, mask the presence of
peptides in LC-MS experiments of untreated urine. Removal of
organic acids is therefore a prerequisite for the analysis of small
urinary peptides by LC-MS (when electrospray is the ion source).
In this protocol, this is accomplished by strong cation exchange
(SCX) extraction. At low pH, peptides are positively charged and
thus bind negatively charged sulfonic acid groups on SCX beads,
while organic acids do not bind and can therefore be aspirated to
waste. After extensive washing of beads to remove loosely bound
molecules, peptides are then recovered from SCX beads using
a solution of high ionic strength and relatively high pH. The
extraction protocol makes use of volatile salts whenever possible
in order to make it more compatible with LC-MS analysis. There
is also a reverse-phase (RP) extraction step prior to SCX, which
is needed to deplete the sample of inorganic salts. This desalting
step reduces the ionic strength of the sample thus making possible
the binding of peptides to the SCX material.
Desalted peptides are then analysed by LC-MS/MS.
Although many types of mass spectrometers may be suitable, in