Waters MassPREP Phosphopeptide Standards - Manual (Page 2)

[ Care and Use ManUal ]
Phosphopeptide Standards 2
III. reCommended usage
There are three sample kits available (#1-3 below). Each kit contains
1 nmol each of four synthetic enolase phosphopeptides - T18 1P
(phosphotyrosine), T19 1P (phosphoserine), T43 1P (phosphothreo-
nine and T43 2P, double phosphoserine).
The sample kits are intended for use in optimizing phosphopeptid
detection in mass spectrometry (MS) or liquid chromatography (LC).
These standards can be used on many instrument systems, including
Waters MALDI micro MX™, Alliance, ZQ™, Q-Tof™, LCT Premier™, and
nanoACQUITY UPLC™ systems. Either UV- or MS- based detection is
possible.
IV. samPle PreParatIon
The following methods are provided as a general guideline. For opti-
mum performance, use of the highest purity commercially available
matrices (Waters MassPREP) and solvents (HPLC grade or better) is
recommended.
Procedure
1) Add 100 μL of water (or 0.1% TFA or 0.1% formic acid) to vial
and vortex to make a 10 pmol/μL solution.
2) Make the desired dilutions in preferred solvent.
3) Prior to LC or MS analysis, use enolase digest to adjust instru-
ment settings for optimum peak shape, resolution and sensitiv-
ity. Calibrate instrument.
4) For MALDI-MS, make a solution of 20 mg/mL 2,5-DHB matrix
in 4:1 (v/v) acetonitrile (ACN):water with 0.2 to 2% phosphoric
acid (PA).
a) For Waters MassPREP matrix containing 10 mg/vial, add
400 μL ACN and 100 μL 2% PA in water for overall
concentration of 0.4% PA.
b) Apply 1 μL (or less) of the sample (~ 1 pmol/μL) onto
a clean MALDI target. Add an equal volume of matrix solu-
tion to the sample solution on the target plate. Dry at room
temperature before submitting target for MALDI analysis.
5) For LC analysis, transfer desired amount of solution to appropri-
ate sample vial and subject to LC-UV or LC-ESI-MS analysis.
a) For separations on ~2 mm diameter columns, the recom-
mended injection volume is 15 μL or more of a solution
10 pmol/μL or more.
b) For separations on columns less than 200 μm in diameter,
the recommended injection volume is 1 μL of a solution
50 fmol/μL or more.
Part Number (PN) MassPREP Product
Information
Suggested Application
186003285 #1 - Phosphopeptide
Standard – Enolase (1
nmol each) Test detection sensitivity and
sample preparation methods
with no ion suppression from
non-phosphorylated peptides
186003286 #2 - Enolase Digest with
Phosphopeptides Mix (1
nmol each, equimolar
mix) Compare phosphopeptide
detection before and after
treatment or enrichment pro-
cedures without conducting a
lengthy digestion procedure
186003287 #3 - Phosphopeptide
Sample Kit – Enolase
(contains PNs
186003285 and
186002325) Conduct control experiments
with phosphopeptides only
and enolase digest only and
make samples with different
phosphopeptide to unmodi-
fied peptide ratios to test
enrichment procedures