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Table 8 Typical problems and troubleshooting in QTAP

From: A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

Problem Possible reasons Solutions
No signal peak is observed for authentic peptides (St or IS) in SRM/MRM analysis. Incorrect LC conditions and/or SRM/MRM parameters (e.g., incorrect m/z of SRM/MRM transition). Ensure that the correct conditions and parameters are used.
Observed m/z is mismatched to theoretical m/z in MS analyzer. Conduct mass calibration.
Decrease in sensitivity of MS analyzer. Clean MS device.
The authentic solution is not injected in autosampler. · Check the remaining volume of solution in the well. Repair if necessary.
· Do not introduce bubbles when applying solution to wells.
Liquid leak in LC-MS/MS system. Determine whether there is liquid leak. Repair if necessary.
Sensitivity of peptide is not high enough. Peptide is not retained in or not eluted from column. Change the target peptide.
No reproducible result for quantitative values. Loss of proteins/peptides during sample preparation due to immature technique. The extent of loss is different between samples. · Need additional practice.
· Add a fixed amount of artificial protein (“monitoring protein”) to every protein sample before sample preparation, and quantify the digested peptides of the artificial protein by LC-MS/MS after sample preparation to evaluate the recovery rate (%) in the sample preparation.
The signal peak is partially occluded by background noise. Use a high-resolution MS analyzer such as a TripleTOF5600.
Inappropriate peak recognition or inappropriate range of calibration curve used for quantification. · Use a basic rule of peak recognition.
· The range of the calibration curve should be adjusted according to the expression level of the target proteins, or the sample should be diluted to be quantified within the linear range of the calibration curve.
Protein expression of target protein is not detected in any tissues or cells. Efficacy of enzyme digestion (LysC and trypsin) is extremely low. Change the target peptide, and avoid the transmembrane region.
Protein expression level is below the limit of quantification in any tissues and cells. · Use a more sensitive MS analyzer or target peptides.
· Purify and concentrate the target protein or peptide.