Waters MassPREP Phosphopeptide Enrichment Kit User Manual
[ Care and Use ManUal ]
Ma s s P R E P™ P H OSP HO P E P T IDE ENRI C HM ENT K IT
I. INT R O DUC T ION
The MassPREP™ Phosphopeptide Enrichment Kit is a tool for selective
enrichment of phosphopeptides from complex proteomic samples. This
kit contains a micro-scale solid-phase extraction (SPE) plate packed with
a unique affinity sorbent. This affinity SPE does not require pre-loading
the sorbent with metal chelation. Also, superior selectivity is achieved
compared to other existing affinity chromatographic methods.
The selectivity of this sorbent towards phosphopeptides is further
improved by adding a unique chemical to the sample before loading
onto the SPE. This chemical (under the trade name, Enhancer) improves
the overall selectivity by displacing the non-phosphorylated peptides
during the sample loading step.
The MassPREP™ Phosphopeptide Enrichment Kit includes the 96-well
micro elution (µElution) plate, enhancer, and a test sample for method
validation. The test sample is a mixture of four synthetic phosphopeptides
mixed with yeast enolase tryptic peptides in 1:1 molar ratio. A general
guideline for kit operation and specific protocols for phosphopeptide
extraction from complex protein digests are provided. The 96-well
micro elution (µElution) platform conveniently uses a vacuum manifold,
for reproducibility and high throughput of enrichment experiments.
CON T ENT S
I. INT RODUCTION
II. MATERIALS INCLUDED IN THE KIT
III. PHOSPHOPEPTIDE AFFINITY ENRICHMENT METHOD
IV. PHOSPHOPEPTIDE ENRICHMENT KIT USAGE GUIDELINES
Part A. How to Use the MassPREP™ Phosphopeptide Enrichment
μElution Plate
Part B. How to Use the MassPREP™ Enolase/Phosphopeptide
Standard as a Test Sample
Part C. How to Apply Enhancer for Highly Complex Samples
Part D. Sample Preparation for Mass Spectrometry Analysis
V. ORDERING INFORMATION
VI. STORAGE AND DISPOSAL OF USED PLATES
[ Care and Use ManUal ]
II. MAT E R I A L S INC L U D E D I N T H E K I T
1. MassPREP™ Phosphopeptide Enrichment µElution plate (part number
186003820). This plate contains 96 wells. Each well is packed
with 2.5 mg of affinity sorbent. The µElution plate is operated by
a vacuum manifold. Related accessories are listed at the end of the
document.
2. MassPREP™ Enhancer (part number 186003821). A total of ten
vials of lyophilized powder are included in the kit (500 mg/vial).
This chemical, termed enhancer, is used to significantly improve the
selectivity of phosphopeptide enrichment by displacing non-phospho-
peptides from the affinity sorbent in the loading step. It is a critical
component of the method.
3. MassPREP™ Enolase/Phosphopeptide Standard (part number 186003286)
contains four synthetic phosphopeptides mixed with yeast enolase
tryptic peptides (Swiss-Prot P00924) in 1:1 molar ratio. The four
synthetic phosphopeptides are modified enolase tryptic peptides that
contain phosphoserine, phosphothreonine, and phosphotyrosine. The
amino acid sequences, phosphorylation sites, and the mass to charge
ratio of the singly and doubly protonated peptide ions are listed
in Table 1. This standard sample can be used as a test sample for
method evaluation before using the customer samples.
III. P H O S P HO P E P T ID E A FF I N I T Y E N RIC H M E NT M E T HO D
Table 2: Solutions Required for the Protocol
Solution Composition of Solution
Condition Solution I Milli-Q® grade water
Condition Solution II 0.2 - 0.5% TFA (v/v) in 80% acetonitrile
Loading Solution (No Enhancer) 0.2 - 0.5% TFA (v/v) in 80% acetonitrile
Loading Solution (with Enhancer) Solubilize the Enhancer with 0.2%-0.5%
TFA in 80% acetonitrile to a final concentration of 50 to 100 mg/mL
Wash Solution I 0.2 - 0.5% TFA in 80% acetonitrile
Wash Solution II Milli-Q® grade water
Elution Solution 100 mM Diammonium Phosphate or
1-2% (v/v) Triethylamine
a. Using highly concentrated enhancer in the sample may result in
loss of phosphopeptides. The recommended concentration is less
than 100 mg/mL.
b. Adding EDTA in the elution solution improves recovery of multiply
phosphorylated peptides, yet interferes with mass spectrometry
analysis. See Section III, D for advices on sample clean up for mass
spectrometry analysis.
Table 1. Amino Acid sequences and m/z ratios of the four synthetic
phosphopeptides
Phosphopeptide
Description
T18_1P NVPL(pY)K 813.39 407.20
T19_1p HLADL(pS)K 863.40 432.21
T43_1p VNQIG(pT)LSESIK 1368.68 684.84
T43_2P VNQIGTL(pS)E(pS)IK 1448.64 724.83
Sequence [M+H] [M+2H]
2
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