The validity o
f using elemental phosphorus standards toaccurately and precisely quanti
fy phosphopeptides
bycapillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements tomaintain sta
ble
31P sensitivity along the reversed-phasegradient are descri
bed. Speci
fically, the use o
f an optimum postcolumn makeup
flow with a de
fined acetonitrilecontent turned out to
be necessary to
bu
ffer the acetonitrile variation o
f the capillary chromatographic eluent andensure plasma sta
bility. Then, a highly pure P-containingstandard (
bis(4-nitro-phenyl) phosphate, BNPP) wasspiked into the samples and used to quanti
fy them withvery low a
bsolute errors (2-4%) and excellent precision(3-6%). The capHPLC-ICPMS method showed excellentlinearity over 3 orders o
f magnitude and provided adequate detection limits (110
fmol, 3.4 pg P). Accuratequanti
fication o
f the phosphopeptides present in a trypticdigest o
f fchars/
beta2.gi
f" BORDER=0 ALIGN="middle">-casein and casein
from
bovine milk was thenattempted. Previously, and in order to
be a
ble to closemass
balances, total P contents, percentages o
f inorganicP present, and recoveries
from the reversed-phase column used in the separation were computed
for eachsample. Quanti
fication using the spiked BNPP
for thedi
fferent phosphopeptides detected matched the expectedvalues well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidatingamino acid sequences, a requisite still necessary totranslate the determined amount o
f P in each chromatographic peak into amount o
f phosphopeptide. The greatpotential o
f these strategies,
based on ICPMS detection,to assess the many procedures proposed and commonlyused
for puri
fication, preconcentration, and/or isolationo
f phosphopeptides in phosphoproteomics studies isdemonstrated using a commercially availa
ble titaniumdioxide (TiO
2) cartridge
for phosphopeptide enrichment
from complex mixtures. Quantitative results o
btainedallow one to assess individual phosphopeptide recoveries
from the TiO
2 cartridge with unsurpassed accuracy. O
fcourse, this in
formation is essential
for relia
ble a
bsolutequanti
fications in phosphoproteomics.