The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.

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Background: Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. Methods: We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. Results: Total run-time including sample cleanup was 8 min. Intra-and interassay analytical variation (CV)

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100 000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.

The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a “Z-spray” type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.

To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).

We present a new variable temperature (VT), high resolution ion mobility (IM) drift tube coupled to a commercial mass spectrometer (MS). Ions are generated in an electrospray ion source with a sampling cone interface and two stacked ring RF guides which transfer ions into the mobility analyzer located prior to a quadrupole time-of-flight mass spectrometer. The drift cell can be operated over a pressure range of 0.5-3 Torr and a temperature range of 150-520 K with applied fields typically between 3 and 14 V cm. This makes the instrument suitable for rotationally averaged collision cross section (CCS) measurements at low E/N ratios where ions are near thermal equilibrium with the buffer gas. Fundamental studies of the effective ion temperatures can be performed at high E/N ratios. An RF ion trap/buncher is located at the beginning of the drift region, which modulates the continuous ion beam into spatially narrow packets. Packets of ions then drift in a linear electric field, which is 50.5 cm long, and are separated according to their mobility in an inert buffer gas. Post-drift, an ion funnel focuses the radially spread pulses of ions into the inlet of a commercial MS platform (Micromass QToF2). We present the novel features of this instrument and results from VT-IM-MS experiments on a range of model systems-IMS CCS standards (Agilent ESI Tune Mix), the monomeric protein Ubiquitin (8.6 kDa), and the tetrameric protein complex Concanavalin A (103 kDa). We evaluate the performance of the instrument by comparing ambient CCS values of model compounds with those found in the literature. Several effects of temperature on collision cross sections and resolution are observed. For small rigid molecules, changes in resolution are consistent with anticipated thermal diffusion effects. Changes in measured CCS for these rigid systems at different temperatures are attributed primarily to the effect of temperature on the long-range attractive interaction. Similar effects are seen for protein ions at low temperatures, although there is also some evidence for structural transitions. By heating the protein ions, their conformational profiles are significantly altered. Very high temperatures narrow the conformational space presented by both Ubiquitin and Concanavalin; it appears that diverse conformational families are "melted" into more homogeneous populations. Because of this conformational heterogeneity, the apparent IMS resolution obtained for proteins at ambient and reduced temperatures is an order of magnitude lower than the expected diffusion limited resolution (R). This supports a hypothesis that the broad CCS features frequently observed for proteins do not correspond to interconverting conformers, but rather to high numbers of intrinsically stable structures.