Quantification challenge: 2 matrices for a complex protein

Usually quantitative analyses of proteins in serum are performed on derived peptides obtained from a peptidic digestion after an immunoaffinity depletion of most abundant protein, to avoid interferences in analyte detection, loss of sensitivity due to surface stickiness of whole proteins, and because serum contains high levels of Alpha 1-antitrypsin that dramatically reduces the efficiency of of serine protease depended digestion steps. In our work, we developed a quantitative analysis on whole serum, without any prior immunodepletion, for a protein with an high isoelectrical point that suffers of bad non-specific binding, by using a commercial platform for solid phase digestion, combined with an immunoaffinity purification on magnetic beads, and by deploying the high resolution and mass accuracy of Orbitrap-XL (Thermo). The protein that we have to analyze is a recombinant form of a native protein naturally present in human body. Health Authorities requested us to clearly identify the recombinant protein and to distinguish it from the native form. This limitation forced us to choose peptides form N-term or the C-term part of the protein that are different in primary structure from the native form. The 8AA N-term peptide is favorable, because has an isoelectrical point below 5 and it is unique. In order to follow and quantify our digested peptide we designed an 8AA peptide in which we modified 1AA. By using the Orbitrap-XL (Thermo), we decided to push the tuning file on charge +2. We monitored the exact mass (0.03Da window) of monoisotopic peak of the peptide. This gave us 300 pg/mL as instrumental buffer sensitivity of the peptide with a very strong linearity. Due to high non-specific binding phenomenon that strongly affect our protein we decided to use a direct digestion of h-serum, since depletion of the serum most abundant proteins was not possible, because we lost our protein during the purification. For this digestion we had to improve the digestion rate with surfactants, that really helped us to increase the yield of peptide. To evaluate the digestion and purify the sample we developed three different methods: SPE on RP that gave us 500 ng/mL as LOD, protein precipitation that gave us 150 ng/mL as LOD, and immunoaffinity protocol with a rabbit polyclonal antibody coated on Protein-G magnetic beads. This last approach gave us 150 pg/mL as LOD. At the moment the methods is proficient and reproducible for concentration above 60 ng/mL. This protocol was successfully applied on other matrices in which protein is present, with results comparable with serum.