Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n=15), while the RSDs of the peak areas were less than 15% (n=15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimensio

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012