Q: If I already removed small contaminants and detergents at the protein level, do I still need to desalt my peptides prior to mass spectrometry analysis?

Yes. We recommend performing additional cleanup after protein digestion to remove any residual salts or partially digested proteins using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852), Pierce C18 Spin Tips (Cat. No. 84850) or an in-line C18 trap column (https://www.thermofisher.com/order/catalog/product/164564-CMD). Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center .

Q: Can I use protein desalting columns for cleanup of peptide samples?

No. Protein desalting columns (e.g., Zeba desalting columns) use size exclusion and have a molecular weight cut-off that is typically too high for peptide samples. Peptide desalting, on the other hand, uses reversed-phase chromatography resins (e.g., C18 resin) to bind peptides for salt removal during washing. Find additional tips, troubleshooting help, and resources within our Protein Dialysis, Desalting, and Concentration Support Center .

Q: How effective is the salt removal using Pierce Peptide Desalting Spin Columns?

With Pierce Peptide Desalting Spin Columns, more than 90% of salt or labeling tags such as TMT tags can be removed. Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center .

Q: What is the reproducibility of Pierce Peptide Desalting Spin Columns from sample to sample?

With Pierce Peptide Desalting Spin Columns, the reproducibility coefficient of variation (CV) is ± 20%. Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center .

Question 1

How do I determine if my sample is compatible and ready for LC-MS?

Accepted Answer

Samples prepared using LC/MS grade reagents are suitable for LC-MS; however, particulates and other small molecules can all interfere with liquid chromatography separation and mass spectrometer source ionization. We recommend visual inspection of samples for particulate matter and using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852), Pierce C18 Spin Tips (Cat. No. 84850) or an in-line C18 trap column (https://www.thermofisher.com/order/catalog/product/164564-CMD) to remove non-volatile salts before MS analysis.

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

Question 2

After TMT reagent labeling, I still see a lot of excess TMT tags in my sample. How should I remove it?

Accepted Answer

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.

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

Question 3

I'm seeing a peak in my mass spectrometry analysis results that seems to always be present in my system. What can I do?

Accepted Answer

The peak most likely represents PEG or a similar contaminant. Clean your MS system and/or perform sample clean-up using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852) or Pierce C18 Resin. In addition, ensure that LC-MS pre-blended solvents are being used (formic acid/water, formic acid/acetonitrile, etc.)

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

Question 4

I cleaned up my peptide sample using C18 or desalting columns, but I lost most of my peptides. What went wrong?

Accepted Answer

Peptides do not bind well to reversed phase resins at neutral pH or in the presence of organic solvents (e.g., acetonitrile). Acidify protein digest samples using formic acid or trifluoroacetic acid (TFA) to pH <3 before desalting. Ensure that samples do not contain organic solvents before and after clean-up by drying them down using a SpeedVac concentrator or equivalent.

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

Question 5

If I already removed small contaminants and detergents at the protein level, do I still need to desalt my peptides prior to mass spectrometry analysis?

Accepted Answer

Yes. We recommend performing additional cleanup after protein digestion to remove any residual salts or partially digested proteins using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852), Pierce C18 Spin Tips (Cat. No. 84850) or an in-line C18 trap column (https://www.thermofisher.com/order/catalog/product/164564-CMD).

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

Question 6

Can I use protein desalting columns for cleanup of peptide samples?

Accepted Answer

No. Protein desalting columns (e.g., Zeba desalting columns) use size exclusion and have a molecular weight cut-off that is typically too high for peptide samples. Peptide desalting, on the other hand, uses reversed-phase chromatography resins (e.g., C18 resin) to bind peptides for salt removal during washing.

Find additional tips, troubleshooting help, and resources within our Protein Dialysis, Desalting, and Concentration Support Center.

Question 7

How effective is the salt removal using Pierce Peptide Desalting Spin Columns?

Accepted Answer

With Pierce Peptide Desalting Spin Columns, more than 90% of salt or labeling tags such as TMT tags can be removed.

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

Question 8

What is the reproducibility of Pierce Peptide Desalting Spin Columns from sample to sample?

Accepted Answer

With Pierce Peptide Desalting Spin Columns, the reproducibility coefficient of variation (CV) is ± 20%.

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

Pierce™ Peptide Desalting Spin Columns, 25 Columns - FAQs