Biology Reference
In-Depth Information
Fig.
2
Concentration versus Peak Area. A dilution series of four HPs was mea-
sured on a Thermo TSQ Vantage QqQ-MS. The ionization effi ciency, and thus the
signal response, is dependent on the amino acid sequence, as can be seen by
the differing slopes of the linear regression lines
knowledge, no robust method exists to predict the signal intensity
(ionization effi ciency) of a given peptide sequence (Fig.
2
). This
can potentially be a major drawback, since the ionization effi ciency
can only be determined experimentally. Subsequently, an other-
wise suitable peptide can exhibit very low signal intensities, and
thus pose analytical diffi culties. Digestion effi ciency, peptide solu-
bility, ionization effi ciency, and matrix effects are accounted for
within the experimental approach, in contrast to the theoretical
approach.
3.3 Number of
Peptides per Protein
Peptides should be chosen from different regions (e.g., the middle
of and close to the C-terminus) of the protein. The more peptides
per protein measured, leading to identical results, the more certainty
about the quality of the results can be assumed. Principally, one pep-
tide per protein should be suffi cient for quantifi cation. Nonetheless,
at least two peptides per protein should be used for the Mass Western,
if possible, due to variability in proteolytic effi ciency and differing
recovery rates. Necessary validation can thus be performed by com-
paring individual peptide results for a given protein.
