Waters MassPREP Enolase Digest with Phosphopeptides Mix - Manual (Page 2)

[ Care and Use ManUal ]
MassPREP Enolase Digest with Phosphopeptides Mix 2
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. 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
Table 2: Enolase Peptides
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.
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/
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.
Peptide Sequence Isotopic mass, [M+H] + [M+2H] 2+
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
Peptide Sequence
Isotopic mass,
[M+H] + [M+2H] 2+
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
Peptide Sequence
Isotopic mass,
[M+H] + [M+2H] 2+
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
2328.0533 1164.5306