Bioanalysis Zone

A current perspective of supercharging reagents and peptide bioanalysis

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The bioanalysis community has been using LC–MS-based approaches for quantifying small molecule drugs and biomarkers extracted from plasma for at least a couple of decades. During this time LC–MS/MS technology has improved significantly, especially the sensitivity of triple quadrupole instruments. Over the past decade, these instruments have become sufficiently sensitive to detect and quantify larger analytes like peptides and small proteins once they have been extracted from complex matrices. However, these analytes are significantly more challenging to work with than small molecules, and the achievable sensitivity using LC–MS/MS instrumentation is hindered by the same mechanism that enables these higher molecular weight analytes to be detected by bench top instruments in the first place. This hindrance is the acquisition of multiple number of charges in the electrospray source – for example, a hypothetical 3500 kDa peptide with a single charge (m/z 3501) will not be visible using any of the commercially available triple quadrupoles. However, the addition of two more positive charges to the peptide generates an m/z of 1167.7, which is within the m/z range of all commercial instruments. The acquisition of charges in the electrospray source is not a well-controlled process, and the hypothetical peptide would likely be present with m/z values of 1751, 1167.7, 876 and 701 corresponding to the 2, 3, 4 and 5+ charged molecules, respectively.

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