Page 1
[ Care and Use ManUal ]
massprep enolase dIgest wIth phosphopeptIdes mIx
I. IntroduCtIon
As a tool for screening sample preparation and sample enrichment
methods for phosphopeptides, Waters offers the MassPREP™ Enolase
Digest with Phosphopeptides Mix. Commonly used phosphorylated
samples are often beta-casein digests or standards which contain
only two phosphopeptides, both modified at serine. The four modified
enolase peptides in the mixture are either singly phosphorylated at
serine, threonine, or tyrosine or doubly phosphorylated at serine.
The enolase digest and phosphopeptides mixture is provided as a
lyophilized solid in a glass vial. T he vials are vacuum-sealed in foil
pouches to ensure protection from light and air.
Contents
I. IntroduCtIon
II. reCommended usage
III. sample prep aratIon
IV. example speCtra
V. storage and staBIlItY
VI. orderIng InFormatIon
MassPREP Enolase Digest with Phosphopeptides Mix 1
Page 2
[ Care and Use ManUal ]
II. reCommended usage
Each vial contains 1 nmol of digested enolase mixed with 1 nmol
each of four synthetic 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) and mixing with Waters MassPREP Digestion Stan-
dard - Enolase. Using this mixture, the user can test phosphopeptide
detection before and after treatment or enrichment procedures
without conducting a lengthy digestion procedure. T he 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 Isotopic mass, [M+H]+ [M+2H]
T18 1P or T18p NVPLpY K 813.3912 407.1995
T19 1P or T19p HLADL pSK 863.4028 432.2053
T43 1P or T43p VNQIG pTLSESIK 1368.6776 684.8428
T43 2P or T43pp VNQIG TLpSEpS IK 1448.6439 724.8259
2+
Isotopic mass,
Peptide Sequence
T38 TAGIQ IVADD LTVTN PK 1755.9492 878.4786
T44 AAQDS FAAGW GVMVS HR 1789.8444 895.4262
T45 SGETE DTFIA DLVVG LR 1821.9234 911.4657
T6 SIVPS GASTG VHEAL EM 1840.9227 920.9654
T51 IEEEL GDNAV FAGEN
FHHGD K
+
[M+H]
2328.0533 1164.5306
[M+2H]
III. sample preparatIon
The following methods are provided as a general guideline. For opti-
mum performance, use of the highest purity commercially available
solvents is recommended (HPLC grade or better).
Procedure
1. Add 100 µL of water or other preferred solvent to the glass vial con-
taining the lyophilized phosphopeptide standard to make a 10 pmol/uL
solution of each phosphopeptide.
2. Vortex the vial for about 30 seconds to ensure that the solid is com-
pletely dissolved.
2+
Table 2: Enolase Peptides
Isotopic mass,
Peptide Sequence
T28 AAGHD GK 655.3163 328.1621
T12 ANIDV K 659.3728 330.1903
T33 NPNSD K 674.3109 337.6594
T10 GVLHA VK 723.4517 362.2298
T3 SVYDS R 726.3422 363.6750
T18 NVPLY K 733.4250 367.2165
T40 IATAI EK 745.4460 373.2269
T19 HLADL SK 783.4367 392.2224
T32 YDLDF K 800.3830 400.6954
T22 TFAEA LR 807.4365 404.2223
T42 AADAL LLK 814.5038 407.7558
T23 IGSEV YHNLK 1159.6111 580.3096
T11 NVNDV IAPAF VK 1286.7011 643.8546
T43 VNQIG TLSES IK 1288.7112 644.8596
T16 LGANA ILGVS LAASR 1412.8225 706.9153
T4 GNPTV EVELT TEK 1416.7224 708.8652
T14 AVDDF LISLD GTANK 1578.8015 789.9048
[M+H]
+
[M+2H]
3. Make the desired number of dilutions in the preferred solvent.
4. Prior to MALDI, use a standard sample (peptide mix or protein digest)
2+
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/
uL) onto a clean MALDI target. Add an equal volume of matrix
solution to the sample on the target plate. Dry at room tempera-
ture 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.
MassPREP Enolase Digest with Phosphopeptides Mix 2
Page 3
[ Care and Use ManUal ]
m/z
600800 1000 1200 14001600 18002000 22002400
%
0
100
%
0
100
807.3
800.2
756.3
808.3
1579.2
1288.9
1159.8
814.3
1057.7
1161.8
1368.9
1756.4
1581.2
1841.4
2442.9
2329.8
756.3
726.1
723.2
807.3
808.3
1413.1
1159.7
814.3
1057.7
1288.9
1841.4
1579.2
1778.4
2442.9
1896.5
DHB with 1% PA
DHB
m/z
1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480
%
0
100
%
0
100
1368.9
1357.9
1417.0
1413.1
1369.9
1370.9
1373.0
1418.0
1449.0
1419.0
1420.0
1451.0
1413.1
1368.9
1357.9
1390.9
1371.0
1398.1
1406.9
1414.1
1417.0
1439.0
1435.1
1419.0
1449.0
1471.0
1461.1
T43 1P
T43 2P
T43 1P
T43 2P
DHB with 1%
PA
DH
B
m/z
810815 820825 830835 840845 850855 860865 870
%
0
100
%
0
100
814.3
809.3
810.3
815.4
816.4
863.3
814.3
809.3
810.2
836.3
829.3
822.2
815.4
816.3
823.2
827.3
835.2
830.3
832.3
863.3
837.3
857.3
842.4
851.3
844.2
859.4
864.3
868.2
870.3
T18 1P
T19 1P
T19 1P
T18 1P
DHB with 1%
PA
DHB
Time
5.00 10.0015.00 20.0025.00 30.0035.00 40.0045.00
5.00 10.0015.00 20.0025.00 30.0035.00 40.0045.00
LCMS
T18 1P
T43 2P
T19 1P
T43 1P
T18 1P
T43 2P T43 1P w/enolase pept ides
T19 1P w/enolase
peptides
MassPREP Phosphopeptide Standard - Enolase
MassPREP Enolase Digest with Phosphopeptides Mix
MassPREP Phosphopeptide Standard - Enolase
MassPREP Enolase Digest with Phosphopeptides Mix
Time
5.00 10.0015.00 20.0025.00 30.0035.00 40.0045.00
5.00 10.0015.00 20.0025. 00 30.0035.00 40.0045.00
LCUV
T18 1P
T43 2P
T19 1P
T43 1P
T18 1P
T43 2P
T43 1P w/enolase pept ides
T19 1P w/enolase
pepti des
ii. For separations on columns less than 200 µm in diameter,
the recommended injection volume is 1 µL of a solution
200 fmol/µL or more.
IV. example speCt ra
The following MALDI TOF MS spectra of MassPREP Enolase Digest
with Phosphopeptides Mix (10 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). Where
indicated, some samples were analyzed using 1% phosphoric acid
(PA) in the solvent mixture used to solubilize the DHB matrix. In
addition, a number of commercially available phosphopeptide
enrichment strategies can be used to selectively improve the signal
intensity of the phosphopeptides.
Figure 1: Enolase Digest with Phosphopeptides, 600 to 2600 Da
Figure 3: Closer view of T18 1P and T19 1P
The following LC-ESI-MS (TIC) and LC-UV (at 210 nm) 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™ C
3 μm, PA – A, 2.1 x 150 mm column. Data
18
from the Waters MassPREP Phosphopeptide Standard – Enolase is
included in the following figures in order to more clearly identify the
phosphopeptide LC peaks.
Figure 2: Closer view of T43 1P and T34 2P
MassPREP Enolase Digest with Phosphopeptides Mix 3
Figure 4: LCMS graph
Figure 5: LCUV graph
Page 4
[ Care and Use ManUal ]
V. storage and staBIlItY
It is recommended that the MassPREP Enolase Digest with
Phosphopeptides Mix 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.
VI. orderIng InFormatIon
Related Product Descriptions Part Number
MassPREP Phosphopeptide Standard - Enolase 186003285
MassPREP Phosphopeptide Sample Kit - Enolase 186003287
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 (1 pk) - for plate storage 290000479
BioSuite C
3 μm, PA – A, 2.1x 150 mm 1860002427
18
Sales Offices:
Austria and European Export
(Central South Eastern Europe,
CIS and Middle East) 431 877 18 07
Australia 2 9933 1777
Belgium 32 2 726 1000
Brazil 55 11 5094 3788
Canada 800 252 4752
China 8621 6495 6999
CIS/Russia +7 495 3367000
Czech Republic 42 02 617 11384
Denmark 45 46 59 8080
Finland +358 9 5659 6288
France (33) 1 30 48 72 00
Germany 49 6196 400600
Hong Kong 852 29 64 1800
Hungary 36 1 350 5086
India and India Subcontinent
91 80 2 837 1900
Ireland 353 1 448 1500
Italy 39 02 274 211
Japan (81) 3 3471 7191
Korea (82) 2 820 2700
Mexico 5255 5200 1860
The Netherlands +31 (0)76-50 87 200
Norway 47 63 84 60 50
Poland (48) 22 833 4400
Puerto Rico 787 747 8445
Singapore 65 6273 1221
Spain 34 93 600 93 00
Sweden 46 8 555 11500
Switzerland 41 62 889 2030
Taiwan 886 2 2543 1898
© 2008 Waters Corporation. Waters, The Science of W hat’s
possible, BioSuite, MALDI micro MX and MassPREP are
trademarks of Waters Corporation
June 2008 715001713 Rev A VW-P DF
MassPREP Enolase Digest with Phosphopeptides Mix 4
United Kingdom 44 208 238 6100
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
Loading...