Abstract
Matrix effect is the alteration of an analyte's concentration-signal response caused by co-existing ion components. With electrospray ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the electrospray ionization droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: we investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E. coli digest matrix (∼2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, nondepleted human plasma matrices (∼10(7) proteome) for six human plasma peptide multiple reaction monitoring assays. Our data set demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p < 0.01), with nonuniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision effects in a low concentration (∼0-250 ng/ml) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition stable isotope dilution calibration method was used. Relative residual error (%) and precision (CV%) by stable isotope dilution were within <20%; however, error in phospholipid-depleted and nondepleted plasma matrices were significantly higher compared with no-matrix (p = 0.006). Finally a novel reverse-polynomial dilution calibration method with and without phospholipid-depletion was compared with stable isotope dilution for relative residual error and precision. Reverse-polynomial dilution techniques extend the Lower Limit of Quantification and reduce error (p = 0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed multiple reaction monitoring assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.
Highlights
The results indicated significant ion suppression when a biological matrix was introduced for all four target peptides (p Ͻ 0.05; Table III) and similar trends experienced by their stable-isotope analog counterparts
Raw ion intensity signals varied greatly for all peptides with the introduction of a complex sample matrix, and was highly peptide-specific: For (AMBP) HHGPTITAK peptide, there was no difference in ion signal between no-matrix and very little matrix (0.001 g/l E. coli) but ion signal was significantly suppressed when a 0.01 g/l or greater concentration of matrix components was present
For the (AMBP) - HHGPTITAK stable-isotope analog, there was high fluctuation of ion signals in complex matrices and no significant difference was noted between no-matrix and any of the comparative matrices
Summary
Target Synthetic Peptide Preparation—Light (crystalline powder) and stable isotope-labeled synthetic AQUA (heavy) peptides for six proteotypic human plasma peptides were purchased from SigmaAldrich (Missouri, CO) at greater than 95% purity (Table I). Labeled forms contained either a C-terminal N15, C13 on Arg, Lys, or internal Leu. All peptides were aliquoted into 1 nM amounts following amino acid analysis. Amino acid analysis was carried out at the Australian Proteome Analysis Facility with all peptides made up in stock amounts to 1 mg/ml with 50% acetonitrile, 5% acetic acid, and 0.1% trifluoroacetic acid (TFA). 20 g amounts of synthetic peptides were reconstituted in 200 l of 20% acetonitrile, 0.1% TFA, put into 10 l aliquots, and dried down. These samples were put through 24 h gas phase hydrolysis with 6 M HCl at 110 °C and analyzed in duplicate using the Waters AccQTag Ultra chemistry on a Waters (Milford, MA) Acquity UPLC. The quantitative values were averaged and used for subsequent analyses
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