Mass Spectrometry Sample Quantitation Support—Troubleshooting

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.

Labeling efficiency can be determined for individual labeled samples by searching for peptides using TMT/TMTpro as a variable (i.e., dynamic) modification peptide amino terminus and lysines. The ratio of peptide to tag by mass (w:w) should be 1:4 to 1:8 for complete labeling of most samples.

Low labeling efficiency can also be caused by improper storing or handling of TMT/TMTpro reagents that leads to hydrolysis of -NHS groups on the tag, rendering the reagents to be less reactive.

We recommend verifying that SILAC media without light lysine (and/or arginine) and dialyzed FBS was used for cell culture. Also, verify that cultured cells are healthy, viable, and actively growing in SILAC media, and free from microbial contamination.

We recommend using Proteome Discoverer Software, MaxQuant Software, or Skyline Software (for targeted analysis) for protein and peptide quantitation.

We recommend reviewing your sample-prep workflow to ensure consistent protein extraction, reduction/alkylation, digestion, and clean-up. We recommend using EasyPep products for high quality, reproducible sample preparation (EasyPep Maxi Sample Prep Kit, EasyPep Mini MS Sample Prep Kit, EasyPep 96 MS Sample Prep Kit). We also recommend quantifying peptides using the Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290) or Pierce Quantitative Coloimetric Peptide Assay (Cat. No. 23275) to ensure that the same amount of peptides are being used for each LC-MS analysis. Poor reproducibility could also be related to the LC-MS system performance which may require recalibration using Pierce Calibration Solutions. System performance can be assessed using protein digest standards such as Pierce HeLa Protein Digest Standard or Pierce TMT11plex Yeast Digest Standard and peptide standards such as Pierce Peptide Retention Time Calibration Mixture or Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix).