Mass Spectrometry Sample Clean-Up Support—Troubleshooting

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.

Some detergents can be removed from peptide samples using Pierce Detergent Removal Resin or HiPPR Detergent Removal Spin Columns in the HIPPR (High Protein and Peptide Recovery) format. However, detergents are more easily removed at the protein level using acetone precipitation, dialysis, or affinity capture using Pierce Detergent Removal ResinHiPPR Detergent Removal Spin Columns before protein digestion.

We recommend checking your mass spectrometry system performance using the Pierce HeLa Protein Digest Standard (Cat. No. 88328) to help determine if the problem is from the sample preparation or the LC-MS system.

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. No. 89851 or 89852) or Pierce C18 Resin. In addition, ensure that LC-MS pre-blended solvents are being used (formic acid/water, formic acid/acetonitrile, etc.)

Extra peptides in the sample may be introduced during digestion from self-cleavage of the protease or contaminate proteases. We recommend using LC-MS grade proteases which are purer and modified to reduce the number of autolytic peaks in the sample.

Your mass spectrometry instrument may require calibration. We recommend calibrating using one of our Pierce Calibration Solutions. Additionally, verify that correct search parameters were used for database searching (e.g., species, enzyme, fragment ions, mass tolerance, etc.).

This indicates poor peptide digestion due to improper pH, or incompatible salts (e.g., urea, guanidine etc.).

Peptide concentration can be measured using our Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290) or Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275).

We recommend using the Pierce HeLa Protein Digest Standard (Cat. No. 88328) to test your sample clean-up method. Use it directly and also as a control, co-treated with the sample to check for any loss of peptides.

We offer EasyPep MS sample preparation kits (EasyPep Maxi Sample Prep Kit, EasyPep Mini MS Sample Prep Kit, EasyPep 96 MS Sample Prep Kit) 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 with extra washes using 5% methanol. The Pierce High pH Reversed-Phase Peptide Fractionation Kit can also be used to remove excess unreacted TMT tags before collecting fractions.