When To Use Relative or Absolute Quantitation Strategies
Experimental bias can influence the decision to use relative or absolute quantitation strategies. One source of bias is the mass spectrometer itself, which has a limited capacity to detect low-abundance peptides in samples with a high dynamic range. Additionally, the limited duty cycle of mass spectrometers restricts the number of collisions per unit of time, which may result in an undersampling of complex proteomic samples. Another source of bias is variation in sample preparation between experiments or individual samples in single experiments. The greater the number of steps between labeling and sample combination, the greater is the risk of introducing experimental bias. For example, during metabolic labeling, proteins are labeled in live animals or cells and the samples are then immediately combined. Because all subsequent sample preparation and analysis is performed with the combined samples, metabolic labeling has the lowest risk of experimental variation. Conversely, samples that are individually processed and analyzed in label-free quantitation strategies have a greater risk of sample variation and experimental bias.
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