Waters MassPREP Phosphopeptide Standard - Manual

[ Care and Use ManUal ]
MassPREP Phosphopeptide Standard – Enolase 
I. IntroductIon
As a tool for screening sample preparation and sample enrichment methods
for phosphopeptides, Waters offers the MassPREP
™ Phosphopeptide Standard
– Enolase. Commonly used phosphorylated samples are often beta-casein
digests or standards which contain only two phosphopeptides, both modified
at serine. The enolase phosphopeptide standard contains four modified enolase
peptides, singly phosphorylated at either serine, threonine, or tyrosine or dou-
bly phosphorylated at serine. The phosphopeptides are packaged as lyophilized
solids in glass vials. The vials are vacuum-sealed in foil pouches to ensure
protection from light and air.
II. recommended usage
Each vial contains  nmol each of four synthetic enolase phosphopeptides
(T8 (P, tyrosine), T9 (P, serine), T43 (P, threonine), T43 (2P, serine)).
This standard was prepared by purifying bulk synthetic peptide from GenScript
Corp. (Piscataway, NJ). Using this standard, the user can test phosphopeptide
detection without competing components as well as add these phosphopep-
tides to any desired mixture. The standard is intended for use in optimizing
phosphopeptide detection in liquid chromatography (UV or MS detection) and
matrix-assisted laser desorption/ionization (MALDI).
Table 1: Synthetic Enolase Phosphopeptides
III. sample preparatIon
The following methods are provided as a general guideline. For optimum
performance, use of the highest purity commercially available solvents is
recommended (HPLC grade or better).
a. Procedure
. Add 00 μL of water or other preferred solvent to the glass vial
containing the lyophilized phosphopeptide standard to make a 0
pmol/µL solution of each phosphopeptide.
2. Vortex the vial for about 30 seconds to ensure that the solid is
completely dissolved.
3. Make the desired number of dilutions in the preferred solvent.
4. Prior to MALDI, use a standard sample (peptide mix or protein
digest) to adjust instrument settings for optimum resolution and
sensitivity for the mass range between m/z 800 and 2000.
a. For MALDI, make a solution of 20 mg/mL 2,5-DHB matrix in
4: (v/v) ACN:water with 0.% TFA.
b. Apply  μL (or less) of the phosphopeptide standard
(~  pmol/µL) onto a clean MALDI target. Add an equal
volume of matrix solution to the sample on the target plate.
Dry at room temperature before submitting target for MALDI
analysis.
5. Prior to HPLC, use enolase digest to adjust instrument settings
for optimum resolution and sensitivity. Adjust parameters so that
peak shape and detector sensitivity are optimized.
a. For HPLC, transfer desired amount of solution to appropriate
sample vial and subject to LC-UV and/or LC-MS analysis.
i. For separations on ~2 mm diameter columns, the recom-
mended injection volume is 5 µL or more of a solution 0
pmol/µL or more.
ii. For separations on columns less than 200 um in diameter,
the recommended injection volume is  µL of a solution
200 fmol/µL or more.
massprep phosphopeptIde standard – enolase
Peptide Sequence Average MW
(g/mol)
Isotopic mass,
[M+H] +
Isotopic mass,
[M+2H]2 +
T8 P or T8p NVPLpYK 82.858 83.392 407.995
T9 P or T9p HLADLpSK 862.875 863.4028 432.2053
T43 P or T43p VNQIGpTLSESIK 368.444 368.6776 684.8428
T43 2P or T43pp VNQIGTLpSEpSIK 448.424 448.6439 724.8259