Waters MassPREP Phosphopeptide Standard User 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 doubly 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 1 nmol each of four synthetic enolase phosphopeptides
(T18 (1P, tyrosine), T19 (1P, serine), T43 (1P, 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
Peptide Sequence Average MW
(g/mol)
T18 1P or T18p NVP LpYK 812.858 813.3912 407.1995
T19 1P or T19p HLA DLpSK 862.875 863.4028 432.2053
T43 1P or T43p VNQIGpTLSESIK 1368.444 1368.6776 684.8428
T43 2P or T43pp VNQIGT LpSEpSIK 1448.424 1448.6439 724.8259
Isotopic mass,
+
[M+H]
Isotopic mass,
[M+2H]2
+
III. sample preparatIon
a. Procedure
1. Add 100 μL of water or other preferred solvent to the glass vial
containing the lyophilized phosphopeptide standard to make a 10
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
4:1 (v/v) ACN:water with 0.1% TFA.
b. Apply 1 μL (or less) of the phosphopeptide standard
(~ 1 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 15 µL or more of a solution 10
pmol/µL or more.
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).
MassPREP Phosphopeptide Standard – Enolase 1
ii. For separations on columns less than 200 um in diameter,
the recommended injection volume is 1 µL of a solution
200 fmol/µL or more.
[ Care and Use ManUal ]
T43
T18
T43
T19
m/z
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
1368.72
813.51
863.52
1448.65
Ti me
12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
AU
0.0
2.0e-2
4.0e-2
6.0e-2
8.0e-2
210
Range: 9.871e-2
Area
17.34
14867
15.69
9416
22.38
11337
24.61
10585
T18 1P
T19
T43
Ti me
10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00
%
0
100
T43
T43 2P
T18 1P
T43
T19
IV. example spectra
The following MALDI TOF MS spectrum of MassPREP Phosphopeptide Standard
– Enolase (2 pmol/sample well) mixed with MassPREP MALDI Matrix - DHB
(20 mg/mL) was processed by baseline subtraction. Data in positive reflectron
mode was obtained on a Waters MALDI micro MX™ (sum of 200 laser shots).
The following LC-ESI-MS in positive mode (top) and LC-UV at 210 nm (bottom)
spectra of the MassPREP Phosphopeptide Standard – Enolase was obtained
using a Waters Alliance® 2795 coupled to a Waters ZQ™ 4000 mass spec-
trometer. 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.
MassPREP Phosphopeptide Standard – Enolase 2
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