Protein Turnover Measurements in Human Serum by Serial Immunoaffinity LC-MS/MS

Abstract

The utility of clinical pulse-chase studies to investigate protein turnover can be extended by serial immunoaffinity enrichment of target proteins. Turnover analysis from serum and subsequently from remaining supernatants provided analytical sensitivity and reproducibility for multiple human target proteins in the same sample set, irrespective of the order of analysis.

Human FcRn Tissue Expression Profile and Half-Life in PBMCs

“…PBMC samples were collected longitudinally over a 36 h period including at time 0, i.e., before administration of the labeled leucine. A protein immunoaffinity enrichment method was required that would not consume the entire sample as the supernatant of each pulse labelling sample can be used for subsequent turnover analysis of other proteins of interest [12]. Cells were lysed and FcRn was immunoprecipitated using an anti-human FcRn antibody.…”

“…An averaged enrichment profile was prepared using mean values at each time point and was used for data fitting and half-life calculation for each subject. Protein synthesis kinetics information was extracted from fitting both the leucine precursor data and peptide data using SAAM II software [12,25]. When applying a first order kinetics relationship between leucine (precursor) and the peptide (product), the synthesis rate constant (k syn ) was derived and an average half-life of 11.1 h was calculated for FcRn in PBMCs of two healthy volunteers, as shown in Table 4.…”

“…Twenty mictroliters of pre-washed streptavidin T1 MyOne Dynabeads (Invitrogen, Carlsbad, CA, USA) were added to each sample and incubated on a mixer at room temperature for 45 min. Automated bead processing steps were described previously [12,16,17]. Briefly, captured proteins were eluted from washed beads with the addition of 140 µL of 30 mM HCl and neutralized with mixing with 30 µL of 1 M Tris HCl pH 8.…”

“…× 15 cm), applying a 10 min gradient from 2% to 45% organic solvent (98% ACN, 0.1% formic acid) with a total run time of 16 min, and analyzed by TSQ-Quantiva™ triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Details of MS settings have been described [12].…”

“…Furthermore, stable isotope-labeled (SIL) leucine pulse and pulse-chase labeling in human subjects followed by protein immunoaffinity and targeted mass spectrometry has been applied for measurement of turnover of target proteins [12,13,14,15]. In this study, we measured the FcRn expression profile in human tissues as well as FcRn half-life in peripheral blood mononuclear cells (PBMCs) collected from a U- 13 C-leucine pulse labeling study in normal human healthy volunteers.…”