|Application of 2D-LC for the traceable quantification of human growth hormone in serum
|Challenges in Quantitative Analysis
|Dr Sophie Inman
Abstract Information :
Recently, there has been a drive to provide higher order reference materials to the clinical community to facilitate reliable & reproducible measurements in relevant settings across spatial and temporal scales. These reference materials must be characterised using metrological (traceable) techniques such as exact matching isotope dilution mass spectrometry. The accurate quantification of proteins such as human growth hormone from biological matrices such as serum encounters a number of challenges. Primarily, serum is a complex mixture of proteins that exist over a wide range of concentrations and in a number of different proteoforms. There are a number of steps to facilitate the traceable quantification of a protein by mass spectrometry. Briefly, isotopically labelled protein or peptides are spiked into the matrix and the intact proteins are digested into peptides. The natural and labelled peptides, unique to the protein measurand, are quantified.
Liquid chromatography coupled to selected reaction monitoring on triple quadrupole instrumentation is highly selective towards the analytes of interest. However, in a complex mixture such as a whole serum digest, there will be a number of peptides with similar chemical properties thus likely to co-elute in a typical separation. These co-eluting peptides could potentially result in ion suppression of the peptides of interest. The additional separation space generated in multi-dimensional chromatography using orthogonal separation methods can be tuned to improve selectivity towards the peptides of interest and thereby improve sensitivity in a complex mixture by reducing ion suppression effects from co-eluting peptides.
Starting with a method which involved two semi-preparative scale off-line chromatographic separations with fraction collection prior to reverse phase LC-MS, we have developed an online 2D-LC method for the detection of six peptide markers of human growth hormone including a peptide that distinguishes between the two most abundant isoforms circulating in the blood stream.
Because we were using a home-built set up, we have been able to make a number of modifications in the method development process. Firstly, we moved from a step gradient in the first dimension with a single trapping column to a gradient elution and two trapping columns to reduce run times. The addition of a divert valve facilitated the change from this fully comprehensive set up to heart-cutting. Furthermore, to improve the sensitivity of the mass spectrometry measurements, a capillary flow pump has been added for the 2D dimension separation.
Although the method still uses one semi-prep scale off-line separation, the manual sample preparation time has significantly reduced from approximately a week from sample digestion to LC-MS analysis to 2.5 days. Furthermore, by reducing the number of off-line separations and resuspension steps, we believe sample recoveries have been improved.