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In-Solution Tryptic Digestion and Guanidination Kit |
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Obtain complete digestion and excellent sequence coverage.
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Accurate identification of proteins and analysis of post-translational modifications by mass spectrometry require accurate and complete protein digestion and peptide modification. The In-Solution Tryptic Digestion and Guanidination kit provides an optimized procedure and reagents for approximately 90 digests of 0.025 - 10 µg of protein.
Trypsin specifically cleaves peptide bonds at the carboxyl side of arginine and lysine residues. The signals from the arginine-containing peptides are generally stronger, due to the more basic side chain. To enhance overall ionization, guanidination is necessary to convert lysines to homoarginines. The guanidination reaction is specific for the amine of lysine but may
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occur minimally at the amine of the peptide's N-terminus. This derivatization leads to an increase in the intensity of the lysine containing peptides and improved sequence coverage overall.
Highlights:
- Optimized — digestion has been optimized to produce complete digests of protein ranging from 0.025 µg - 10 µg. Guanidination reaction has been optimized to produce little to no N-terminal modification
- Convenient — kit includes reagents for digestion, reduction, alkylation and guanidination.
- Quick — protein can be digested and guanidinated all in one day
Summary of Procedure:
- Denature/reduce 95°C, 5 minutes
- Alkylation in the dark at room temperature, 1 hour
- Digestion 37°C, 5 hours; alternatively overnight
- Guanidination 65°C, 12 minutes
- Stop Reaction
Figure A.
Figure B.
Comparison of a guanidinated digest with a non-guanidinated control demonstrates improved ionization and sequence coverage after guanidination. Figure A: MS spectra of digested BSA (100 ng). Figure B: MS spectra of digested BSA (100 ng) with guanidination. Guanidination results in a mass shift of +42 m/z for each lysine in a peptide (#) and (*) indicated two lysine-containing peptides with and without guanidination for demonstration purposes.
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# Peaks
No Guanidination Control |
# Peaks
Guanidinated Digest |
| Lysozyme |
6 |
11 |
| Myoglobin |
7 |
11 |
| BSA |
17 |
27 |
Table 1: Increasing Sequence Coverage. Tryptic peptides with a terminal arginine ionize in MS analysis preferentially over lysine terminating peptides often resulting in lost data. To reduce this bias and to enhance overall ionization, guanidination is necessary to convert lysines to homoarginines. This modification leads to an increase in the intensity of the lysine containing peptides and improved sequence coverage. Significantly greater numbers of peptides are identified from a variety of test proteins after guanidination. Peptides identified determined based on scanning from 600-2000m/z.
Related Products
In-Gel Tryptic Digestion Kit
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Ordering Information
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 Bulk quantities: Inquire  Certificate
of Analysis  Product
Instructions  MSDS |
| Product # |
Description |
Pkg. Size |
Files |
Price |
| 89895 |
In-Solution Tryptic Digestion and Guanidination Kit
| Sufficient reagents for preparing 90 digests. | | Kit contains: | | Trypsin, Modified | 20 µg | | Trypsin Storage Solution | 50 µl | | Ammonium Bicarbonate | 50 mg | | No-Weigh DTT | 1 tube | | Iodoacetamide (IAA) | 500 mg | | O-Methylisourea Hemisulfate Salt (OMI) | 400 mg | | Ammonium Hydroxide | 1 ml | |
Kit |
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$278.00
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