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MassPREP Phosphopeptide Standard – Enolase 1

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 dou-

bly 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

III. sample preparatIon

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).

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.

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.

massprep phosphopeptIde standard – enolase

Peptide Sequence Average MW

(g/mol)

Isotopic mass,

[M+H]

Isotopic mass,

[M+2H]2

T18 1P or T18p NVPLpYK 812.858 813.3912 407.1995

T19 1P or T19p HLADLpSK 862.875 863.4028 432.2053

T43 1P or T43p VNQIGpTLSESIK 1368.444 1368.6776 684.8428

T43 2P or T43pp VNQIGTLpSEpSIK 1448.424 1448.6439 724.8259

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Podsumowanie treści

Strona 1 - [ Care and Use ManUal ]

[ Care and Use ManUal ]MassPREP Phosphopeptide Standard – Enolase 1I. IntroductIonAs a tool for screening sample preparation and sample enrichment

Strona 2

[ Care and Use ManUal ]MassPREP Phosphopeptide Standard – Enolase 2IV. example spectraThe following MALDI TOF MS spectrum of MassPREP Phosphopeptide

Strona 3

[ Care and Use ManUal ]Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.comMassPREP Phosphop

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