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AccuMAP™ Low pH Protein Digestion Kit

Suppress Artificial Deamidation and Disulfide Bond Scrambling in Protein Sample Prep

  • Sample preparation completed in 4.5–5 hours
  • Highly reproducible digestion results
  • Use for reduced and nonreduced proteins

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Catalog number selected: VA1040

$ 780.00
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AccuMAP™ Low pH Protein Digestion Kit
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$ 780.00
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Suppress Nonenzymatic PTMs During Protein Sample Prep

Nonenzymatic posttranslational modifications (PTMs) spontaneously occur in biotherapeutic proteins during manufacturing and storage. The major nonenzymatic PTMs are deamidation, disulfide bond scrambling and oxidation. These modifications can affect efficacy and stability of biotherapeutic proteins and are the subject of careful monitoring.

Nonenzymatic PTMs can also be induced during protein sample preparation for peptide mapping and can compromise analysis. Major causes of nonenzymatic PTMs induced during sample preparation include alkaline pH and impurities that have protein oxidizing activity.

Suppress Artificial Nonenzymatic PTMs

The AccuMAP™ Low pH Protein Digestion Kits suppress artificial nonenzymatic PTMs, as sample preparation is performed at low pH under either reducing or nonreducing conditions.

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Schematic diagram of sample preparation by digestion performed with AccuMAP™ Low pH Protein Digestion Kit.

Protein Digestion at Low pH

Trypsin and other proteases commonly used in peptide mapping sample preparation favor alkaline pH in order to efficiently digest proteins. To avoid artificial nonenzymatic PTMs induced at these conditions, we developed trypsin digestion at low pH. To restore trypsin cleavage efficiency at lysine sites we supplemented trypsin with a special, low pH-resistant recombinant Lys-C (rLys-C) protease. By supplementing trypsin with low pH resistant rLys-C we achieved efficient tryptic digestion at low pH.

13961MA-W
Efficient digestion with modified trypsin and AccuMAP™ Low pH Resistant rLys-C at low pH. Rituximab was digested according to a conventional protocol (at pH 8) or with the AccuMAP™ Low pH Protein Digestion Kit at low pH and analyzed by LC/MS. The data show that digestion at low pH was comparable to digestion at conventional conditions.

Suppression of Deamidation and Disulfide Bond Scrambling

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13962MA-W-b-2

Complete suppression of deamidation and disulfide bond scrambling in IgG digested with AccuMAP™ Low pH Protein Digestion Kit. Figure shows extracted ion chromatogram of a GLEWIGAIYPGNGDTSYNQK peptide from Rituximab antibody digested at conventional conditions (pH 8) and at low pH (with AccuMAP™ Low pH Protein Digestion Kit). The data show suppression of deamidation in the upper panel and the complete suppression of disulfide bond scrambling in the lower panel.

Minimal Baseline Noise in Tryptic Digests

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13963MA-W-b-2

UV-HPLC chromatograms of Panitumumab digests. Panitumumab was predigested with AccuMAP™ Low pH Resistant rLys-C, then the reaction was diluted and digestion was completed by incubation with trypsin for 3 hours (upper panel) or overnight (lower panel). In this experiment we used a 1:5 trypsin:protein ratio. Note the accumulation of baseline noise for the overnight digest shown in the lower panel.

Low-pH Mapping Method Using rLys-C and Modified Trypsin

This publication illustrates the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifacts, and achieve efficient mAb digestion equivalent to the conventional tryptic peptide-mapping method under alkaline conditions.

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping. (2019)  J. Pharm Sci. Jul 30. pii: S0022-3549(19)30485-X. doi: 10.1016/j.xphs.2019.07.019

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Characterization and Quantification of Succinimide Using Peptide Mapping Under Low-pH Conditions

Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis. Succinimide has long been known to be an intermediate product, but its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site.

Characterization and Quantification of Succinimide Using Peptide Mapping Under Low-pH Conditions and Hydrophobic Interaction Chromatography (2019) Anal. Biochem. 566, 151–59.

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Specifications

You are viewing: VA1040 Change Configuration

What's in the box?

Item Part # SizeConcentrationAvailable Separately

AccuMAP™ Modified Trypsin Solution

V528C 2 × 60μg0.5μg/μl View Product

AccuMAP™ Denaturing Solution

VA100A 1 × 1ml View Product

AccuMAP™ 10X Low pH Reaction Buffer

VA101A 1 × 1ml View Product

AccuMAP™ 100X Oxidation Suppressant

VA102A 1 × 50μl View Product

AccuMAP™ Low pH Resistant rLys-C Solution

VA103A 2 × 60μg200μg/ml View Product

TCEP

VB100A 3 × 15mg View Product

Iodoacetamide

VB101A 3 × 15mg View Product

NEM

VB102A 2 × 15mg

SDS

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Download SDSPDF (272 KB) – English (United States)

Certificate of Analysis

See all certificates for VA1040

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

Specifications

You are viewing: VA1050 Change Configuration

What's in the box?

Item Part # SizeConcentrationAvailable Separately

AccuMAP™ Modified Trypsin Solution

V528D 3 × 400μg View Product

AccuMAP™ Denaturing Solution

VA100B 2 × 5ml View Product

AccuMAP™ 10X Low pH Reaction Buffer

VA101B 2 × 5ml View Product

AccuMAP™ 100X Oxidation Suppressant

VA102B 2 × 250μl View Product

AccuMAP™ Low pH Resistant rLys-C Solution

VA103B 5 × 240μg200μg/ml View Product

TCEP

VB100A 5 × 15mg View Product

Iodoacetamide

VB101A 5 × 15mg View Product

NEM

VB102A 4 × 15mg

SDS

Choose language:

Download SDSPDF (272 KB) – English (United States)

Certificate of Analysis

See all certificates for VA1050

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

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