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Peptide Enrichment for Mass Spectrometry
Post digestion, peptide enrichment is often necessary to detect peptides from low-abundant proteins and/or for reducing sample complexity for proteomic analysis.
There are several strategies that can be deployed to increase target protein identifications: utilize post-translational modification (PTM) specific affinity binding for phosphopeptide enrichment, or use of a probe, such as the novel Thermo Scientific ActivX™ probes, to target and enrich specific enzyme classes, including kinases, GTPases, and serine hydrolases.
- Comprehensive—reagents and kits for multiple peptide enrichment strategies
- Optimized—kits are designed to maximize peptide yield and increase target protein ID
- Convenient—easy-to-use spin columns, tips or magnetic formats enable more rapid processing of peptide samples
- Flexible—kits can be used with different sample types (cultured mammalian cells, tissues, etc.)
- Validated—all products have been fully tested and processed samples have been analyzed using Thermo Scientific Mass Spectrometers
Choose the right peptide enrichment products for your applications
Phosphorylation is a protein modification important to biological functions such as cell signaling, growth, differentiation and division, and programmed cell death; however, phosphopeptides have high hydrophilicity and are low in abundance, resulting in poor chromatography, ionization and fragmentation. Phosphopeptide enrichment is therefore crucial to successful MS analysis.
Metal oxide affinity chromatography (MOAC) and immobilized metal ion affinity chromatography (IMAC) are two of the most common phosphopeptide enrichment methods. Each method isolates different phosphopeptide sequences, so we offer a variety of columns, spin tips, and kits to support different research applications and goals.
Phosphopeptide enrichment kits
 |  |  |  |  | |
|---|---|---|---|---|---|
| Magnetic Phospohopeptide Enrichment Kit | Fe-NTA Phosphopeptide Enrichment Kit | TiO2 Phosphopeptide Enrichment and Clean Up Kit | Graphite Spin Column | TiO2 Phosphopeptide Enrichment Spin Tips | |
| Binding/labeling mechanism | Metal chelate affinity to phosphate groups | Metal chelate affinity to phosphate groups | Metal oxide affinity to phosphate groups | Graphite affinity to hydrophilic peptides | Metal oxide affinity to phosphate groups |
| Enrichment strategy | Fe-NTA magnetic beads | IMAC-Agarose resin | Spherical porous TiO2 | Graphite spin column | Spherical porous TiO2 |
| Loading capacity/rxn* | 25–1,000 µg | 0.5–5 mg | 0.5–3 mg | 100 µg | 300–1,000 µg |
| Format | Magnetic beads | Spin column | Tip | Spin column | Spin tips |
| Processing time | <45 minutes | 45–60 min | 45–60 min | 10–20 min | 35–45 min |
| Order now | Order now | Order now | Order now | Order now |
*Loading capacity per reaction is based on a typical mammalian cultured cell protein digest sample
The Thermo Scientific Pierce Kinase Enrichment kits utilize an ActivX™ ATP or ADP probe to covalently label the active site of ATPases, including kinases, chaperones and metabolic enzymes. These probes feature an amine-reactive nucleotide analog and a desthiobiotin (biotin analog) tag that facilitates selective labeling of lysines in the kinase active site and then subsequent enrichment and recovery of labeled protein. These features allow identification and profiling of target enzyme classes across samples or assessment of the specificity and affinity of enzyme inhibitors.
For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested to peptides. Only the desthiobiotin-labeled, active-site peptides are enriched for analysis by LC-MS/MS for identification of global inhibitor targets and off-targets.
Kinase enrichment kits
 | ||
|---|---|---|
| Kinase Enrichment Kit with ATP Probe | Add Kinase Enrichment Kit with ADP probe | |
| Binding/labeling mechanism | Biotinylated ATP analog to active site lysine | Biotinylated ADP analog to active site lysine |
| Enrichment strategy | Immobilized streptavidin agarose resin | Immobilized streptavidin agarose resin |
| Loading capacity/rxn* | Lyophilized | |
| Format | Spin column | Spin column |
| # of elution fractions | 1 | 1 |
| Processing time | 6.5 hr + digestion | 6.5 hr + digestion |
| Order now | Order now |
Phosphorylation is a protein modification important to biological functions such as cell signaling, growth, differentiation and division, and programmed cell death; however, phosphopeptides have high hydrophilicity and are low in abundance, resulting in poor chromatography, ionization and fragmentation. Phosphopeptide enrichment is therefore crucial to successful MS analysis.
Metal oxide affinity chromatography (MOAC) and immobilized metal ion affinity chromatography (IMAC) are two of the most common phosphopeptide enrichment methods. Each method isolates different phosphopeptide sequences, so we offer a variety of columns, spin tips, and kits to support different research applications and goals.
Phosphopeptide enrichment kits
| | | | | |
|---|---|---|---|---|---|
| Magnetic Phospohopeptide Enrichment Kit | Fe-NTA Phosphopeptide Enrichment Kit | TiO2 Phosphopeptide Enrichment and Clean Up Kit | Graphite Spin Column | TiO2 Phosphopeptide Enrichment Spin Tips | |
| Binding/labeling mechanism | Metal chelate affinity to phosphate groups | Metal chelate affinity to phosphate groups | Metal oxide affinity to phosphate groups | Graphite affinity to hydrophilic peptides | Metal oxide affinity to phosphate groups |
| Enrichment strategy | Fe-NTA magnetic beads | IMAC-Agarose resin | Spherical porous TiO2 | Graphite spin column | Spherical porous TiO2 |
| Loading capacity/rxn* | 25–1,000 µg | 0.5–5 mg | 0.5–3 mg | 100 µg | 300–1,000 µg |
| Format | Magnetic beads | Spin column | Tip | Spin column | Spin tips |
| Processing time | <45 minutes | 45–60 min | 45–60 min | 10–20 min | 35–45 min |
| Order now | Order now | Order now | Order now | Order now |
*Loading capacity per reaction is based on a typical mammalian cultured cell protein digest sample
The Thermo Scientific Pierce Kinase Enrichment kits utilize an ActivX™ ATP or ADP probe to covalently label the active site of ATPases, including kinases, chaperones and metabolic enzymes. These probes feature an amine-reactive nucleotide analog and a desthiobiotin (biotin analog) tag that facilitates selective labeling of lysines in the kinase active site and then subsequent enrichment and recovery of labeled protein. These features allow identification and profiling of target enzyme classes across samples or assessment of the specificity and affinity of enzyme inhibitors.
For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested to peptides. Only the desthiobiotin-labeled, active-site peptides are enriched for analysis by LC-MS/MS for identification of global inhibitor targets and off-targets.
Kinase enrichment kits
| ||
|---|---|---|
| Kinase Enrichment Kit with ATP Probe | Add Kinase Enrichment Kit with ADP probe | |
| Binding/labeling mechanism | Biotinylated ATP analog to active site lysine | Biotinylated ADP analog to active site lysine |
| Enrichment strategy | Immobilized streptavidin agarose resin | Immobilized streptavidin agarose resin |
| Loading capacity/rxn* | Lyophilized | |
| Format | Spin column | Spin column |
| # of elution fractions | 1 | 1 |
| Processing time | 6.5 hr + digestion | 6.5 hr + digestion |
| Order now | Order now |
Other products for peptide enrichment & fractionation for mass spec
Featured data
Figure 1. High-Select Bead phosphopeptide enrichment at microscale. Nocodazole-treated HeLa cells were prepared and quantitated prior to enrichment with High-Select Fe-NTA Magnetic Beads. Bead: sample ratio remained constant (1:50 w/w) while peptide sample input varied from 10–150 µg. Phosphopeptide IDs were equivalent from 25–150 µg, with a loss of 27% phosphopeptides at 10 µg (A). Phosphospecificity remained >90% for all peptide enrichment input amounts (B).
Number of Phosphates per Peptide
| 1 | 2 | 3 | 4 | 5 | 6 | Total | |
|---|---|---|---|---|---|---|---|
| TiO2 | 492 | 103 | 8 | 4 | 0 | 1 | 608 |
| Fe-NTA IMAC | 234 | 34 | 216 | 3 | 1 | 0 | 488 |
| Overlap | 155 | 0 | 1 | 0 | 0 | 0 | 156 |
Selective enrichment of singly and multiply phosphorylated phosphopeptides with TiO2 and Fe-NTA IMAC. Average phosphopeptide enrichment results from duplicate experiments showing the number of phosphopeptides containing one or more phosphate per peptide enriched using either method. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0 and analyzed with Microsoft™ Excel™ Software.
Figure 1. High-Select Bead phosphopeptide enrichment at microscale. Nocodazole-treated HeLa cells were prepared and quantitated prior to enrichment with High-Select Fe-NTA Magnetic Beads. Bead: sample ratio remained constant (1:50 w/w) while peptide sample input varied from 10–150 µg. Phosphopeptide IDs were equivalent from 25–150 µg, with a loss of 27% phosphopeptides at 10 µg (A). Phosphospecificity remained >90% for all peptide enrichment input amounts (B).
Number of Phosphates per Peptide
| 1 | 2 | 3 | 4 | 5 | 6 | Total | |
|---|---|---|---|---|---|---|---|
| TiO2 | 492 | 103 | 8 | 4 | 0 | 1 | 608 |
| Fe-NTA IMAC | 234 | 34 | 216 | 3 | 1 | 0 | 488 |
| Overlap | 155 | 0 | 1 | 0 | 0 | 0 | 156 |
Selective enrichment of singly and multiply phosphorylated phosphopeptides with TiO2 and Fe-NTA IMAC. Average phosphopeptide enrichment results from duplicate experiments showing the number of phosphopeptides containing one or more phosphate per peptide enriched using either method. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0 and analyzed with Microsoft™ Excel™ Software.
Resources
Stylesheet for Classic Wide Template adjustments
For Research Use Only. Not for use in diagnostic procedures.