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massPreP PhosPhoPePtIde standards
I. IntroduCtIon
Phosphorylated proteins and peptides are difficult to detect and
characterize by mass spectrometry due to their low abundance and
low ionization efficiency. A set of standard samples can aid in devel-
oping methods for optimized phosphopeptide detection. As a tool
for screening sample preparation and sample enrichment methods
for phosphopeptides, Waters offers MassPREP™ Phosphopeptide
Standards containing synthetic phosphopeptides based on modified
peptides from tryptic yeast enolase digest (SwissProt P00924).
Commonly used phosphorylated samples are ß-casein digests or
standards which contain only two phosphopeptides, both modified
atserine. T he four modified enolase peptides in the mixture are either
singly phosphorylated at serine, threonine, or tyrosine or doubly
phosphorylated at serine.
II. synthetIC enolase PhosPhoPePtIdes
Peptide Sequence Isotopic mass,
[M+2H]2+
[M+H]+
Contents
I. IntroduCtIon
II. synthetIC enolase PhosPhoPePtIdes
III. reCommended usage
IV. samPle PreParatIon
V. examPle sPeCtra
VI. storage and stabIlIty
VII. related aCCessorIes
T18 1P NVPLpY K 813.3912 407.1995
T19 1P HLADL pSK 863.4028 432.2053
T43 1P VNQIG pTLSES IK 1368.6776 684.8428
T43 2P VNQIG TLpSEpS IK 1448.6439 724.8259
Phosphopeptide Standards 1
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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).
Part Number (PN) MassPREP Product
Information
186003285 #1 - Phosphopeptide
Standard – Enolase (1
nmol each)
186003286 #2 - Enolase Digest with
Phosphopeptides Mix (1
nmol each, equimolar
mix)
186003287 #3 - Phosphopeptide
Sample Kit – Enolase
(contains PNs
186003285 and
186002325)
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.
Suggested Application
Test detection sensitivity and
sample preparation methods
with no ion suppression from
non-phosphorylated peptides
Compare phosphopeptide
detection before and after
treatment or enrichment procedures without conducting a
lengthy digestion procedure
Conduct control experiments
with phosphopeptides only
and enolase digest only and
make samples with different
phosphopeptide to unmodified peptide ratios to test
enrichment procedures
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.
Phosphopeptide Standards 2
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.
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%
0
100
%
0
100
Ma ssPREP™Enolase Digest with Phosphopeptides Mix, 2 pmol
Ma ssPREP™Phosphopeptide
Standard-Enolase, 1pmol
733.4 775 .1
813.4
814.4
815.4
863.4
1359.1
1368.7
1370.7
1448.7
1450.6
T18 1P
T19 1P
T43 1P
T43 2P
726.3
756.4
757.4
807.4
813.3
863.3
1057.5
1161.5
1288.6
1368.6
1412.7
1488.5
864.4
TOF LD+
5.23e3
TOF LD+
6.04e3
%
0
100
%
0
100
Ma ssPREP™Enolase Digest with Phosphopeptides Mix, 2pmol
Ma ssPREP
™
Phosphopeptide
Standard-Enolase, 1pmol
733.4 775 .1
813.4
814.4
815.4
863.4
1359.1
1368.7
1370.7
1448.7
1450.6
T18 1P
T19 1P
T43 1P
T43 2P
726.3
756.4
757.4
807.4
813.3
863.3
1057.5
1161.5
1288.6
1368.6
1412.7
1488.5
864.4
TOF LD+
5.23e3
TOF LD+
6.04e3
Time
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
%
0
100
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
%
0
100
T19 1P
T18 1P
T43 2P
T43 1P
T18 1P
T43 2P
T19 1P and otherpeptides
T143 1P and other peptides
MassPREP™Phosphopeptide
Standard-Enolase, 600pmol
MassPREP
™
EnolaseDigest with
Phosphopeptides Mix, 300pmol
V. EXAMPLE SPECTRA
The following MALDI-MS spectra (positive reflectron) of Phosphopep-
tide Standard – Enolase (top) and Enolase Digest with Phosphopep-
tides Mix (bottom) mixed with MassPREP DHB matrix were obtained
on a Waters MALDI micro MX. Each spectrum (200 laser shots) was
processed by baseline subtraction.
The following postive mode LC-ESI-MS (TIC) spectra were obtained
using a Waters Alliance 2795 coupled to a Waters ZQ 4000 mass
spectrometer. A 20 pmol/μL solution in 0.02% trifluoroacetic acid
in water was prepared and 30 μL was injected on a Waters BioSuite™
C18 3 μm, PA – A, 2.1 x 150 mm column.
VII. RELATED ACCESSORIES
Related Product Descriptions Part Number
MassPREP Digestion Standard Enolase (1 vial) 186002325
MassPREP Calibration Mix – MALDI Reflectron (1 vial) 186002819
MassPREP MALDI Matrix CHCA (5 vials/1 pk) 186002331
MassPREP MALDI Matrix DHB (5 vials/1 pk) 186002333
MassPREP MALDI Matrix THAP (5 vials/1 pk) 186002335
MALDI Plate Box - for plate storage 290000479
BioSuite C18 3μm, PA – A, 2.1 x 150 mm 186002427
VI. STORAGE AND STABILITY
It is recommended that the MassPREP™ Phosphopeptide Standards
vial remain in the foil pouch (in the dark) at room temperature until
use. Once reconstituted in appropriate solvents, the solution can be
used for one week when stored in the dark at 4 °C. For longer term
storage, the solution should be aliquoted into microcentrifuge tubes
and stored at –20 °C.
Phosphopeptide Standards 3
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©2008 Waters Cor poration, Waters, Alliance, BioSuite, T he
Science of W hat’s Possible, ZQ, Q-Tof, LCT P remier, MALDI Micro
MX, and MassPREP are trademarks of Waters Corporation.
June 2008 715001814 Rev A KK-PDF
Phosphopeptide Standards 4
Waters Corporation
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Milford, MA 01757 U.S.A.
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