Search

Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range, and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate with those observed by ESI-MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase, the extent of charging. This region incorporates properties (e.g., basicities) intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging ("supercharging") such as m-NBA, sulfolane, and 3-nitrobenzonitrile increase analyte charge from "denaturing" and "native" solvent systems. It is suggested that additives' Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte's solution ionization, excess spray charge is bestowed on evaporating ions carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte).

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Monoclonal antibodies (mAbs) form a major class of biologics and recently biosimilars and biobetters are being added to the growing inventory of therapeutics. In-depth characterization of mAbs at various stages [...]

Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of α-syn. In vitro, CLR01 inhibited the assembly of α-syn into β-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. α-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when α-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of α-syn toxicity (α-syn-ZF), which expresses human, wild-type α-syn in neurons. α-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed α-syn aggregation in neurons, and reduced α-syn-induced apoptosis. α-Syn expression was found to inhibit the ubiquitin proteasome system in α-syn-ZF neurons, resulting in further accumulation of α-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson's disease and other synucleinopathies. [ABSTRACT FROM AUTHOR]