When I quantitate my mass spec peptide sample with the Pierce Quantitative Colorimetric Peptide Assay, I get different results than when I use the Pierce Quantitative Fluorometric Peptide Assay. Which is best to use for the most accurate quantitation?
Since the different peptide assays use different chemistries to measure peptides, they may result in different results. Interfering compounds are the most common source of background and inaccurate measurements. Please note that the fluorometric peptide assay is not recommended for peptides which have been modified using TMT reagents.
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.
How can I determine peptide yield in my mass spectrometry samples after sample clean-up?
Peptide yield can be measured using the Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275) or the Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290). The choice of peptide assay depends on the sample type and composition of the sample buffer. The fluorometric peptide assay cannot be used to measure peptides with chemically modified amines such as acetylated peptides or TMT-labeled protein digests. The colorimetric assay can measure a wider range of samples but is not as sensitive as the fluorometric assay, requiring more sample for accurate detection. Finally, both assays are susceptible to interfering compounds in the sample or buffer which should be avoided or removed for best results.
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.
I have good peptide identification numbers but the variation between sample replicates is high. What do you recommend to improve sample reproducibility?
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).
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.
I have varying amounts of peptide in my samples. Is there a way to determine peptide yield after sample clean-up?
Peptide concentration can be measured using our Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290) or Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275).
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.
I have varying amounts of peptide in my mass spectrometry analysis samples. Is there a way I can determine peptide yield after sample clean-up?
You can measure peptide concentration using our Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275) or Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290).
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.