Relative serum protein quantification based upon ICPL and 2D-LC-MS identifies potential frailty biomarkers in elderly patients

This study shows the ability of the ICPL and nano-HPLC-MS/MS to perform relative quantification and identification of serum protein biomarkers.
Frailty is a geriatric syndrome that is commonly associated with the decline in multisystemic reserve, cognition and sensory capabilities. It is negatively influencing the outcome of a disease prolonging the patient’s recovery. The discrepancy between the actual and the biological age brings the uncertainty of predicting frailty in a given individual. This study is addressing the problem of finding suitable biomarkers that bear the ability to objectively predict frailty in elderly patients. It furthermore provides a robust method for reliable relative quantification of serumproteins.
Serum samples used in this study were divided into six groups regarding the patient’s disease (hip-fracture, infection and cardiac decompensation) and frailty status (frail or robust). The individual sera were pooled and a volume of 20 µL was depleted of high abundantproteins. After labeling with ICPL (isotope coded protein label), serum proteins were fractionated according to their respective pI (0-3, 4-7 and 8-12). The samples were further subjected to tryptic digestion followed by the treatment with the Glu-C enzyme. The peptides were analyzed on the 2D-nano-HPLC system (Ulimate 3000®) using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The HPLC system was connected on-line with the electrospray ion-trap mass spectrometer Esquire HCT ultra®.
The relative protein quantification using ICPL and mass spectrometry allowed for comparison of six patient groups with respect to a standard sample. The latter represented a group of healthy old subjects. This technique allowed for the detection of approx. 200 proteins, whereas about 50 % of those contained the ICPL label and could therefore be quantified. The identified proteins covered 3 – 4 orders of magnitude ofprotein concentration in human serum. Several proteins displayed a significant modulation allowing for some preliminary conclusions to be drawn. At this point it can be stated that significantly elevated levels of C-reactive protein (factor 12) and alpha-1-antichimotrypsin (f. 4) proved to be potentially good indicators of frailty. Increased concentrations of alpha-1-microglobulin (f. 4) and alpha-2HS-glycoprotein (f. 2) have been found in the robust patients, whereas no significant concentration alteration could be detected in the frail groups. These results refer to the acute phase response since the samples were collected immediately after patient hospitalization. Current investigations addressing the later sampling times should shed more light on the suitability of these markers to predict frailty in elderly patients. At this stage it is obvious that although several markers are found to be in common for all the frail or robust patients, the disease status additionally complicates the biomarker signature. Therefore a more individualized approach should also be considered, where depending on the age and clinical findings a more defined group of markers should be selected to address the problem of frailty.