The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.

Today, proteomics is an exciting approach to discover potential biomarkers of different disorders. One challenge with proteomics experiments is the wide concentration range of proteins in various tissues and body fluids. The most abundant component in human body fluids, human serum albumin (HSA), is present at concentrations corresponding to approximately 50% of the total protein content in, e.g., plasma and cerebrospinal fluid (CSF). If this component could be selectively removed, then the chances of observing lower-abundance component of clinical interest would be greatly improved. There are today several approaches of varying specificity available for depletion. In this study, the properties of two commercially available kits, for the removal of HSA and HSA and immunoglobulin G (IgG), respectively, were compared, and the benefits of using depletion steps prior to on-line LC-FTICR MS were evaluated. Both methods were applied on plasma and CSF. To our knowledge, these are the first results reported for CSF. Also, the combination with electrospray LC-FTICR MS is novel. The proportion of depleted HSA and IgG was estimated using global labeling markers for peptide quantification. Both depletion-methods provided a significant reduction of HSA, and the identification of lower abundant components was clearly facilitated. A higher proportion of HSA was removed using the affinity-based removal kit, and consequently more proteins could be identified using this approach.

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.