Q: What type of samples can I use with the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit?

The High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit are compatible with desalted and detergent-free protein digests or lyophilized peptide fractions. Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Q: What is the storage buffer composition for the resin slurry in the columns within the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Cat. No. A32992)?

The resin slurry is in 5% acetic acid. Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center .

Q: What is the composition of the Binding/Wash Buffer in the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit?

Here is the composition of the Binding/Wash Buffer in the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit: 0.1 % trifluoroacetic acid (TFA), 80% acetonitrile (ACN). Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Q: I have poor phosphopeptide enrichment specificity using either the High-Select TiO2 Phosphopeptide Enrichment Kit or the High-Select Fe-NTA Phosphopeptide Enrichment Kit. What should I do?

Here are some tips: - Check the expiration date in order to assure that all components are still good. - Keep TiO2 tips protected from light during storage. - Make sure that you are using the correct buffer component for resuspension of your sample, and that the sample is very thoroughly dried and resuspended well. - When incubating peptide samples with the Fe-NTA kit, ensure that you avoid end-over-end rotation or vortexing; instead gently tap the column to disperse the resin. - When removing plugs from columns, be sure to avoid pushing liquid back into the column from the plug reservoir. - After elution, do not store the phosphopeptides in the elution buffer but immediately proceed to drying the sample before storing at -80 degrees C until mass spec analysis. Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Q: After phosphopeptide enrichment using the High-Select Fe-NTA Phosphopeptide Enrichment Kit or High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit, I see a yellow/slight brownish color material in my eluate or after drying my samples. Is this normal?

The yellow/slight brownish color is indicative of iron which has been released from the column during elution along with the phosphopeptides. After acidification of the elution buffer or resuspension of dried peptides in 0.1% formic acid, the material can be removed by centrifugation of the sample at 16,000 x g for 30 sec and transferring the supernatant into a new container for LC/MS injection or peptide quantification. Alternatively, the material can be removed using Pierce Peptide Desalting Spin Columns, Pierce C18 Spin Tips or an in-line C18 trap column. Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Q: I only have 200 ug of protein digest sample. Is this enough for phosphopeptide enrichment using Fe-NTA?

We recommend using at least 0.5-3 mg of clean (i.e., C18 desalted) and dried protein digest for optimal results, with an anticipated yield of 1-3%. Using lower amounts of protein digest sample is possible; however, the phosphopeptide yield will be significantly lower with potentially more non-phosphopeptides present after enrichment, and extra care must be taken to not lose the small amount of sample remaining. Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Question 1

Should I desalt my peptide samples before enrichment using the High Select Phosphopeptide Enrichment Kits?

Accepted Answer

Yes, we do recommend desalting peptide samples before enrichment when using High-Select Fe-NTA and TiO2 phosphopeptide enrichment kits. This helps ensure optimal results by removing detergents and salts that can interfere with the enrichment process.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 2

In the SMOAC protocol (https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/10/High-SelectTM-SMOAC-Protocol.pdf), can I enrich with High-Select Fe-NTA kit first?

Accepted Answer

No. It is important to enrich with the TiO2 kit (Cat. No. A32993) first. Afterwards, the flow-through and wash fractions from this enrichment can be processed with the Fe-NTA kit (Cat. No. A32992). If this order is reversed (that is, Fe-NTA before TiO2), there will be 2 consequences as follows: 1. There will not be any significant additional recovery of peptides (maybe just a few more peptides). 2. There will be no enrichment for the multiple phosphorylated peptides, so those would be lost.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Question 3

Why do you offer two phosphopeptide enrichment kits: the High-Select TiO2 kit (Cat. No. A32993) and the High-Select Fe-NTA kit (Cat. No. A32993)?

Accepted Answer

The two phosphopeptides enrichment kits, Fe-NTA and TiO2, enrich a complementary set of phosphopeptides. Our R&D has developed a Sequential enrichment of Metal Oxide Affinity Chromatography (see https://assets.thermofisher.com/TFS-Assets/CMD/posters/PO-65032-SMOAC-Phosphoproteomics-Peptides-ASMS2017-PO65032-EN.pdf and https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/10/High-SelectTM-SMOAC-Protocol.pdf?CID=bid_mic_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_bid_ts_mbr_24065_Social_LAB) where flow-through and wash fractions from TiO2 enrichment were combined and subjected to Fe-NTA enrichment. This sequential enrichment provides impressive coverage of phosphoproteomes.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Question 4

What are the differences between the old Fe-NTA kit (Cat. No. 88300) and the new High-Select Fe-NTA kit (Cat. No. A32992)?

Accepted Answer

There are four differences between the Fe-NTA kit (Cat. No. 88300) and the new High-Select Fe-NTA kit (Cat. No. A32992) kit as follows: 1. The selectivity - the ratio of number of phosphopeptides over total peptides - was significantly improved to 99% with Cat. No. A32992, because the reagents were extensively optimized for the phosphopeptide selection. 2. The phosphopeptide yield was also increased to 33 µg based on quantitative colorimetric peptide assay (Cat. No. 23275). 3. The reagent is a pre-formulated format, so mixing reagent to prepare the working solution from stock solutions provided in the old kit (Cat. No. 88300) is not necessary, so it is easier to handle. 4. The enrichment protocol is optimized and streamlined, which means there are many fewer steps than with Cat. No. 88300. Thus, it takes <45 min to finish the entire protocol compared to 2 hours with the old kit (Cat. No. 88300).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Question 5

What type of samples can I use with the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit?

Accepted Answer

The High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit are compatible with desalted and detergent-free protein digests or lyophilized peptide fractions.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 6

What is the storage buffer composition for the resin slurry in the columns within the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Cat. No. A32992)?

Accepted Answer

The resin slurry is in 5% acetic acid.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Question 7

What is the composition of the Binding/Wash Buffer in the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit?

Accepted Answer

Here is the composition of the Binding/Wash Buffer in the High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit: 0.1 % trifluoroacetic acid (TFA), 80% acetonitrile (ACN).

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 8

I have poor phosphopeptide enrichment specificity using either the High-Select TiO2 Phosphopeptide Enrichment Kit or the High-Select Fe-NTA Phosphopeptide Enrichment Kit.  What should I do?

Accepted Answer

Here are some tips:
- Check the expiration date in order to assure that all components are still good.
- Keep TiO2 tips protected from light during storage.
- Make sure that you are using the correct buffer component for resuspension of your sample, and that the sample is very thoroughly dried and resuspended well.
- When incubating peptide samples with the Fe-NTA kit, ensure that you avoid end-over-end rotation or vortexing; instead gently tap the column to disperse the resin.
- When removing plugs from columns, be sure to avoid pushing liquid back into the column from the plug reservoir.
- After elution, do not store the phosphopeptides in the elution buffer but immediately proceed to drying the sample before storing at -80 degrees C until mass spec analysis.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 9

After phosphopeptide enrichment using the High-Select Fe-NTA Phosphopeptide Enrichment Kit or High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit, I see a yellow/slight brownish color material in my eluate or after drying my samples. Is this normal?

Accepted Answer

The yellow/slight brownish color is indicative of iron which has been released from the column during elution along with the phosphopeptides. After acidification of the elution buffer or resuspension of dried peptides in 0.1% formic acid, the material can be removed by centrifugation of the sample at 16,000 x g for 30 sec and transferring the supernatant into a new container for LC/MS injection or peptide quantification. Alternatively, the material can be removed using Pierce Peptide Desalting Spin Columns, Pierce C18 Spin Tips or an in-line C18 trap column.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 10

I only have 200 ug of protein digest sample. Is this enough for phosphopeptide enrichment using Fe-NTA?

Accepted Answer

We recommend using at least 0.5-3 mg of clean (i.e., C18 desalted) and dried protein digest for optimal results, with an anticipated yield of 1-3%. Using lower amounts of protein digest sample is possible; however, the phosphopeptide yield will be significantly lower with potentially more non-phosphopeptides present after enrichment, and extra care must be taken to not lose the small amount of sample remaining.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 11

I use the Sequential Enrichment from Metal Oxide Affinity Chromatography (SMOAC) method to enrich phosphopeptides in my mass spec sample. Is it normal for TiO2 enrichment to leave a pellet of non-volatile material in the flow-through sample after drying?

Accepted Answer

Yes, the buffer used for TiO2 column binding contains a non-volatile acid which appears in the sample tube upon drying. The residual material does not interfere with subsequent Fe-NTA enrichment and is removed during/before LC-MS analysis.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Question 12

I want to enrich my mass spec sample for phosphopeptides. Should I use the High-Select TiO2 Phosphopeptide Enrichment Kit, High-Select Fe-NTA Phosphopeptide Enrichment Kit, or both?

Accepted Answer

Each kit has different selectivity for different phosphopeptides. The High-Select TiO2 Phosphopeptide Enrichment Kit enriches more multiply phosphorylated peptides. The High-Select Fe-NTA Phosphopeptide Enrichment Kit enriches more monophosphorylated peptides. In addition, the High-Select Fe-NTA Phosphopeptide Enrichment Kit has slightly higher phosphopeptide specificity and yield than the TiO2 kit. For the best enrichment for all types of phosphorylated peptides, we recommend using our Sequential Enrichment from Metal Oxide Affinity Chromatography (SMOAC) method (https://assets.thermofisher.com/TFS-Assets/CMD/posters/PO-65032-SMOAC-Phosphoproteomics-Peptides-ASMS2017-PO65032-EN.pdf), which utilizes both kits sequentially.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

High-Select™ Fe-NTA Phosphopeptide Enrichment Kit, 30 Reactions - FAQs