alyte. This relationship was not maintained for two of the peptide analytes (Glycinin 1 and Glycinin 2) which were present in such large quantities that matching the signal would require 3 and 2 pmol respectively per injection. For these peptides, a 10:1 (endogenous: AQUA™) ratio was attained, which was still within the pre-determined linear range.
Lastly, our strategy assumed that monitoring one proteotypic peptide is sufficient to represent the whole protein. This assumption is mostly an assumption of complete protein digestion. Although digestion conditions were monitored by Coomassie SDS-PAGE to ensure all proteins were digested to peptides, this approach is not as quantitative as MRM. Ideally, if a protein is subjected to proteolysis using trypsin, the entire protein will be reduced to peptides by cleavage at all Lys and Arg residues. However, this is not necessarily the case. Studies have shown that a number of exceptions exist, which prevent cleavage, or prevent complete cleavage. For example, trypsin was perceived to not cleave at a Lys or Arg when a Pro immediately follows it (Keil, 1992). This rule has been questioned recently. A group using peptide identification data from both low and high mass accuracy proteomics experiments have shown that the prevalence of cuts at the Lys or Arg residues preceding Pro indicates they are not preferred by trypsin but are in fact possible (Rodriguez et al., 2007). The presence of a double cleavage site can also cause issues. If two Lys or Arg residues are immediately adjacent or within a few residues, there is the possibility of incomplete cleavage at any one of the residues. Furthermore, accuracy of quantification is affected by chemistry. Depending on when the internal standards are added (e.g., immediately before, versus immediately after the digestion step), chemical modifications could alter the peptide pool, potentially altering the mass of the endogenous peptide and thereby preventing its measurement during MRM. In our experience, peptide loss due to breakdown or modification is highly dependent on the peptide sequence, with certain combinations of amino acids being more prone to internal or external chemistry. For all of these reasons, using multiple peptides is likely to provide more accurate results. However, with all of the cleavage and chemistry rules considered, most allergens that we wanted to measure have only a single peptide suitable for quantification.
It has been shown that 50–70% of total seed protein is contributed by the storage proteins glycinin and β-conglycinin with their individual percentages varying drastically between studies (Wolf et al., 1961; Saio, 1969; Murphy and Resurreccion, 1984). In our study, cumulative allergen content was lower than expected, with totals approaching 25% (w/w). We believe this is caused by several factors, including fractional quantitation of all paralogous protein forms and alternative protease cleavage sites.
Quantitation of the β-conglycinin fraction was by far the most incomplete which is largely because peptides for β-conglycinin subunits α′ and β were not analyzed in this study, thereby vastly under-representing the β-conglycinin fraction. Secondly, the peptide used for β-conglycinin α may be under represented due to the presence of an “illegitimate” cleavage site (Keil, 1992) that may in fact be partially cleaved in vitro (Rodriguez et al., 2007). Because this peptide is quantifiable, it is obviously not completely cleaved, and therefore may still be useful for quantification if the AQUA™ peptides are spiked prior to digestion, assuming trypsin is capable of stoichiometrically cleaving this small synthetic peptide. Although this observation would affect the absolute levels of this protein, it should not influence relative levels as the protein would likely be equally processed in vitro. The glycinin fraction was the most prevalent in this study, approaching an additive total of nearly 200 μg/mg (∼20%, w/w). This value is still lower than values provided in the literature (ca. 40%), however a large portion of this