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Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit

Catalog No. PI84868
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Catalog No. PI84868 Supplier Thermo Scientific™ Supplier No. 84868
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Description

Increase protein identification from LC/MS analysis through orthogonal peptide fractionation of complex peptide samples using Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit.

In order to enable deep proteome sequencing, it is often necessary to reduce the sample complexity by fractionation in an orthogonal dimension prior to LC/MS analysis. The Pierce High pH Reversed-Phase Peptide Fractionation Kit utilizes high pH reversed-phase chromatography to separate peptides by hydrophobicity, and provides excellent orthogonality to low pH reversed-phase LC-MS gradients. The kit has been designed to improve protein identification through the use of a proprietary reversed-phase resin in an easy-to-use spin column format with a high-pH fractionation protocol.

Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit increases protein identification from LC/MS analysis through orthogonal peptide fractionation of complex peptide samples.

  • Easy-to-use - resin provided in single-use spin column format
  • Improved protein identifications - protein identifications increased by ≥50% when compared to unfractionated samples
  • Reproducible - elution profiles and fractional resolution vary by less than 20%
  • Optimized - robust procedure for maximal protein identification and peptide recovery while minimizing fractional overlap
  • Compatible - reagents validated with a variety of complex samples, including TMT™-labeled peptides
In order to enable deep proteome sequencing, it is often necessary to reduce the sample complexity by fractionation in an orthogonal dimension prior to LC/MS analysis. The Pierce High pH Reversed-Phase Peptide Fractionation Kit utilizes high pH reversed-phase chromatography to separate peptides by hydrophobicity and provides excellent orthogonality to low pH reversed-phase LC-MS gradients.
The kit has been designed to improve protein identification through the use of a proprietary reversed-phase resin in an easy-to-use spin column format with a high-pH fractionation protocol. In contrast to strong cation exchange (SCX) fractionation, the high-pH reversed-phase fractions do not require an additional desalting step before LC/MS analysis.
The High pH Reversed-phase Peptide Fractionation Kit includes a high pH buffer (0.1% triethylamine) and twelve spin columns containing pH-resistant reversed-phase resin. Each reversed-phase fractionation spin column enables fractionation of 10 to 100μg of peptide sample using a microcentrifuge.
Native, phosphorylated, Tandem Mass Tag™ (TMT)-labeled, and other complex peptide mixture samples can be fractionated using this kit. Combining the search results generated by the individual fractions helps improve protein sequence coverage and increases the number of identified proteins relative to unfractionated samples.
Applications:
  • Reducing sample complexity to identify targets of interest
  • Conducting systems biology studies
  • Reducing sample complexity to improve quantitation studies

Specifications

For Use With (Equipment) Microcentrifuge
Product Type Peptide Fractionation Kit
Product Line Pierce
Content And Storage Reversed-phase fractionation spin columns, 12 columns
Triethylamine (0.1%), 100 mL

Store in refrigerator (2–8°C).
Starting Material Peptides, Protease-digested Protein
Final Product Type Peptides
Detection Method Mass Spectrometry
Format Kit
Workflow Step Fractionation
Quantity 12 Reactions

Frequently Asked Questions (FAQs)

Can I purchase the Reversed-Phase Fractionation Spin Columns from the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) separately?

No, the spin columns from this kit cannot be purchased separately. However, if you are only using these columns to remove salts from your peptide samples, you can purchase the Pierce Peptide Desalting Spin Columns (Cat. No. 89851, 89852), as an alternative.
How much starting material is needed to fractionate my samples using the Pierce High pH Reversed Phase Fractionation Kit (Cat. No. 84868)?

A minimum of 100 µg of protein as starting material from the digestion step is needed for fractionation using eight fractions. Less starting material would be needed if fewer fractions are used. If peptides are being measured, the range of peptide to be loaded on these columns is 10 to 100 µg.

I want to fractionate a complex mass spectrometry sample, but my sample is different from the one described in the manual for the Pierce High pH Reversed-Phase Peptide Fractionation Kit. Should I use a customized fractionation gradient?

Recommended gradients are available for typtic protein digest samples with or without TMT labeling. Use of other chemical labels, performing selective peptide enrichment, or using a different digestion enzyme other than trypsin may affect the elution profile of the peptides during fractionation. For the best sample peptide coverage, an ideal elution profile would result in relatively equal amounts of peptides in each fraction.
After running the Pierce TMT11plex Yeast Digest Standard, I observe low signal to noise ratio and poor quantitation accuracy for the Tandem Mass Tag (TMT) reporter ions. What do you recommend?

Here are some recommendations:

- Clean and recalibrate the mass spectrometry instrument using one of our Pierce Calibration Solutions (https://www.thermofisher.com/search/browse/category/us/en/600654).
- Verify settings for liquid chromatography (LC) acquisition methods.
- Fractionate TMT-labeled samples using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) to reduce sample complexity.
I have a lower number of peptide/protein identification in my combined TMT-labeled samples compared to the unlabeled sample. What can I do?

We recommend fractionating the sample using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868), to reduce the sample complexity before LC-MS analysis and thus increase the number of quantifiable peptides/proteins for multiplexed samples.
After TMT reagent labeling, I still see a lot of excess TMT tags in my sample. How should I remove it?

We offer EasyPep MS sample preparation kits (EasyPep Maxi Sample Prep Kit (Cat. No. A45734), EasyPep Mini MS Sample Prep Kit (Cat. No. A40006), EasyPep 96 MS Sample Prep Kit (Cat. No. A45733)) that contain wash buffers specifically formulated to clean up TMT-labeled protein digests. To remove excess TMT reagent from samples prepared using other sample preparation methods, we recommend using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852) with extra washes using 5% methanol. The Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) can also be used to remove excess unreacted TMT tags before collecting fractions.