| Popis: |
Procollagen III amino-terminal propeptide (P-III-NP), a biomarker for detecting growth hormone administration, is currently measured by immunoassays in the absence of international reference material. The main aim of this research is to demonstrate whether liquid chromatography tandem mass spectrometry (LC-MS) can be used to quantify P-III-NP in blood. Low serum concentration (1-5 ng/mL, 25-125 pM) and medium protein size of P-III-NP (~42 kDa) complicates method development, in that there is a requirement for very sensitive LC-MS methods. To meet this, a digest approach with trypsin has been adopted. By LC-HRAMS (high resolution accurate MS in tandem mode) analysis of digested bovine P-III-NP material, post translational modifications (PTMs) of some amino acid residues have been identified through the partial de novo sequencing of peptides. Once assessed for stability, peptides T1 and T5 were synthesized and used to develop sensitive nano- and micro-flow LC-MS methods on a highly sensitive triple quadrupole MS. By both approaches, adequate sensitivity of peptides (with no matrix) has been achieved to accommodate analysis of P-III-NP at basal levels. However, despite the sensitivity of these developed methods, the presence of albumin (HSA, 35-50 mg/mL, 5.3-7.75 x 10-1 mM) in the sample matrix prevents analysis by LC-MS. Through investigation, it has been determined that HSA concentrations >20 μg/mL results in the complete ion suppression of P-III-NP peptides (100 pM) in the MS. Hence, HSA depletion ≥ 99.6 % is necessary to produce samples suitable for LC-MS. Investigations into common proteomic HSA depletion methods have shown that double depletion methods, i.e. protein precipitation (ppt) then molecular weight cut off (MWCO) filters or double MWCO, should be effective in depleting HSA sufficiently from serum. However, further investigations have highlighted that the co-precipitation of P-III-NP with HSA and the poor reproducibility of P-III-NP recovery from MWCO limit the application of these approaches for preparing our serum samples for LC-MS. With the recent (limited) release of an ELISA immunoassay kit targeting P-III-NP, immunocapture of serum P-III-NP prior to trypsin digestion and LC-MS has enabled the identification of peptides from endogenous sources. Using the T5 peptide, the hP-III-NP concentration in a normal human serum sample was semi-quantitatively determined as ~2 ng/mL. For absolute quantification, however, suitable internal standards are needed. Isotopically heavy labelled hT1 and T5 peptide variants have been suggested, for which synthesis and verification has commenced and is presented. |
