Waters MassPREP Enolase Digest with Phosphopeptides Mix User Manual

[ 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

[ 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