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The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.
View the protocol on BioSupplyNet.com. |
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Selected Protocol from "Proteins and Proteomics: A Laboratory Manual," David R. Goodlett, Eugene C. Yi, and Philippe Mottay.
View the protocol "Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion Affinity Media and Direct Analysis by MALDI-TOF-MS" at BioSupplynet.com
Leesa J. Deterding, Jenny M. Cutalo, and Kenneth B. Tomer
ABSTRACT
Phosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe3+, Ni2+, and Ga3+. This technique can be used with either on-line or off-line MS analysis. However, elution of the phosphopeptides from the metal ion column prior to MS analysis can result in sample loss. Affinity-bound analytes, including phosphopeptides, can be directly analyzed by MALDI-MS without prior elution from the affinity media. Also, consecutive enzymatic reactions, such as phosphatase or carboxypeptidase Y digestion, can be carried out on affinity-bound peptides. When the affinity-bound phosphopeptides are treated with phosphatase, the number of phosphorylation sites can be determined based on the observation of 80-Da (or multiples of 80 Da) mass shifts in the MALDI-MS of the reaction mixture. Carboxypeptidase Y treatment of the affinity-bound phosphopeptides can also be used to cleave amino acids from the carboxyl terminus, with subsequent direct analysis of the enzymatic products by MALDI-MS to locate the phosphorylation sites on the bound phosphopeptides. This protocol details the preparation and use of Fe3+ or Ga3+ metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.
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Buy this book, Z700428
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| Products Available for this Protocol |
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Acetic acid |
A6283 |
Acetic acid, ReagentPlus®, ≥99% |
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| Ammonium bicarbonate |
A6141 |
Ammonium bicarbonate, ReagentPlus®, ≥99.0% |
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| Calf intestinal alkaline phosphatase |
79385 |
Phosphatase, Alkaline from bovine intestinal mucosa, BioChemika, solution (clear), >10000 U/mL |
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| EDTA |
E5134 |
Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99% |
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| Carboxypeptidase Y |
C3888 |
Carboxypeptidase Y from baker's yeast (S. cerevisiae), lyophilized powder, ≥50 units/mg protein |
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| Ferric chloride (FeCl3) |
F7134 |
Iron(III) chloride |
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| Gallium chloride (GaCl3) |
427128 |
Gallium(III) chloride, anhydrous, beads, -10 mesh, 99.999+ % |
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| Ethanol |
E7148 |
Ethanol, for molecular biology, 190 proof |
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| H2O |
W4502 |
Water, Molecular Biology Reagent |
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| Formic acid |
F0507 |
Formic acid, reagent grade, ≥95% |
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| Sodium citrate |
S1804 |
Sodium citrate, meets USP testing specifications |
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| TPCK-modified trypsin (sequence-grade) |
93630 |
Trypsin, TPCK treated, from bovine pancreas, BioChemika, ~7500 units/mg |
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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