Atomic Force Microscopy Thermally-Assisted Microsampling with Atmospheric Pressure Temperature Ramped Thermal Desorption/Ionization-Mass Spectrometry Analysis.

The use of atomic force microscopy controlled nanothermal analysis probes for reproducible spatially resolved thermally assisted sampling of micrometer-sized areas (ca. 11 × 17 µm wide × 2.4 µm deep) from relatively low number-average molecular weight (Mn < 3000) polydisperse thin films of poly(2-vinylpyridine) (P2VP) is presented. Following sampling, the nanothermal analysis probes were moved up from the surface and the probe temperature ramped to liberate the sampled materials into the gas phase for atmospheric pressure chemical ionization and mass spectrometric analysis. The procedure and mechanism for material pickup, the sampling reproducibility and sampling size are discussed, and the oligomer distribution information available from slow temperature ramps versus ballistic temperature jumps is presented. For the Mn = 970 P2VP, the Mn and polydispersity index determined from the mass spectrometric data were in line with both the label values from the sample supplier and the value calculated from the simple infusion of a solution of polymer into the commercial atmospheric pressure chemical ionization source on this mass spectrometer. With a P2VP sample of higher Mn (Mn = 2070 and 2970), intact oligomers were still observed (as high as m/z 2793 corresponding to the 26-mer), but a significant abundance of thermolysis products were also observed. In addition, the capability for confident identification of the individual oligomers by slowly ramping the probe temperature and collecting data-dependent tandem mass spectra was also demonstrated. The material type limits to the current sampling and analysis approach as well as possible improvements in nanothermal analysis probe design to enable smaller area sampling and to enable controlled temperature ramps beyond the present upper limit of about 415 °C are also discussed.

Multistage Mass Spectrometry of Phospholipids using Collision-Induced Dissociation (CID) and Metastable Atom-Activated Dissociation (MAD).

We herein demonstrate an approach to gas phase ion manipulation that provides MS3-level CID spectra of phospholipid radical cations that are almost independent of the original charging adduct ions. In the MS2 He-MAD spectra of the protonated, sodiated and potassiated adducts of POPC, the different adducts induce different primary fragmentation pathways and provide significantly different spectra, as is commonly observed by other activation methods. In separate experiments, the even-electron adduct ions ([M+H]+, [M+Na]+, [M+K]+) of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) were first converted to radical cations [POPC]+• by using helium metastable atom-activated dissociation (He-MAD) to eject the charging adduct ions, then exposed to low-energy collision induced dissociation (CID) to induce extensive fragmentation along the acyl chains. Such charge-remote fragmentation is generally inaccessible through low-energy CID of the even-electron precursor ions. The combination of He-MAD and CID provides radical-induced spectra that show very major similarities and only minor differences, and therefore overcomes major differences in chemistry that are otherwise observed by the original adducting species. Collisional activation of even-electron [POPC+H]+ required higher CID amplitudes than odd-electron [POPC]+• to effect fragmentation-as expected-and the latter provided fragments within the acyl chains that were influenced by the double bond position.

Electroactive Polymer-Based Spray Ionization for Direct Mass Spectrometric Analysis.

Electrochemically deposited electroactive polymer (EAP) films were investigated for their potential to enhance the performance of ambient ionization mass spectrometry (MS). Several EAPs of varying hydrophobicity were evaluated, including the superhydrophobic polymer poly[3,4-(2-dodecylethylenedioxy)thiophene] (PEDOT-C12). The EAPs were electropolymerized onto indium tin oxide-coated glass, placed in front of the inlet of a mass spectrometer, and charged to 3.5-4.5 kV. Analyte solutions were then applied to the surface, initiating ionization events. Analytes including peptides and small molecule pharmaceuticals were studied in 0.1% formic acid in methanol/water ("spray solvent") as well as in synthetic biological fluid matrices, using both EAP spray ionization (EAPSI) and paper spray ionization (PSI). Each EAPSI analysis required as little as 0.1 µL of solution, and the resulting sprays were stable and reproducible. The sensitivity, limit of detection (LOD), and limit of quantification (LOQ) were evaluated using bradykinin, cannabinol, and cannabidiol, which were prepared in pure solvents, artificial urine, and artificial saliva. The limits of detection and quantitation for EAPSI were improved relative to PSI by 1-2 orders of magnitude for analytes prepared in methanol/water and on the same order of magnitude as PSI for analytes prepared in artificial saliva and urine. This EAP-based spray ionization technique offers possibilities for rapid MS analysis with small sample sizes, high accuracy, and miniaturization of MS instruments.

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry.

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.

Forensic Mass Spectrometry.

Developments in forensic mass spectrometry tend to follow, rather than lead, the developments in other disciplines. Examples of techniques having forensic potential born independently of forensic applications include ambient ionization, imaging mass spectrometry, isotope ratio mass spectrometry, portable mass spectrometers, and hyphenated chromatography-mass spectrometry instruments, to name a few. Forensic science has the potential to benefit enormously from developments that are funded by other means, if only the infrastructure and personnel existed to adopt, validate, and implement the new technologies into casework. Perhaps one unique area in which forensic science is at the cutting edge is in the area of chemometrics and the determination of likelihood ratios for the evaluation of the weight of evidence. Such statistical techniques have been developed most extensively for ignitable-liquid residue analyses and isotope ratio analysis. This review attempts to capture the trends, motivating forces, and likely impact of developing areas of forensic mass spectrometry, with the caveat that none of this research is likely to have any real impact in the forensic community unless: (a) The instruments developed are turned into robust black boxes with red and green lights for positives and negatives, respectively, or (b) there are PhD graduates in the workforce who can help adopt these sophisticated techniques.

Performance evaluation of a Loeb-Eiber mass filter at 1 Torr.

The Loeb-Eiber mass filter is best operated at relatively high pressures-such as 1 Torr-where collisional dampening of ions up to the mass filter thermalizes the ions' kinetic energy, which is a requirement for effective filtering. The inter-electrode gaps of ~8 µm require rf amplitudes on the order of 0-5 V p-p at approximately 50 MHz to achieve mass filtering up to m/z 40. Mass filtering between the 25-µm diameter wires, therefore, takes place on time frames less than the collision frequency at ~1 Torr. The low power and high pressure capabilities of the Loeb-Eiber mass filter make it ideally suited for miniaturization, where power and space are a premium. In the present work, a Loeb-Eiber mass filter was constructed using commercial silicon-on-insulator (SOI) microfabrication techniques. Ions transmitting through the chip-based Loeb-Eiber mass filter were characterized in real time using a traditional linear quadrupole mass analyzer in series with the Loeb-Eiber mass filter. The new hybrid instrument has enabled us to verify several important claims regarding the operation of the Loeb-Eiber mass filter: (1) that ions can be effectively filtered at ~1 Torr, (2) that for ions of a fixed mass-to-charge ratio, the ion transmission current decreases linearly with increasing rf amplitude on the Loeb-Eiber mass filter, (3) that the cutoff voltage at which all ions of a particular m/z value are effectively blocked is linearly related to mass-to-charge, and (4) that square waveforms can filter ions more effectively than sinusoidal waveforms for a given peak-to-peak rf amplitude.

Charge transfer dissociation (CTD) mass spectrometry of peptide cations using kiloelectronvolt helium cations.

A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from--or charge exchange with--the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H](+) precursor of Substance P gives product ions such as [M+H](2+•) and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C-C(α) cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions.

Nanomanipulation-coupled nanospray mass spectrometry applied to the extraction and analysis of trace analytes found on fibers.

This article presents the novel instrumentation of nanomanipulation coupled to nanospray ionization-mass spectrometry, which is used to directly probe trace analytes found on individual fibers. The low detection limits and sample volumes associated with nanospray ionization-mass spectrometry make it the ideal instrument to implement for trace analysis. Nanospray ionization-mass spectrometry, coupled with the nanomanipulator, allows for the direct probing of trace particulates on fibers. The technique is demonstrated by dissolving an electrostatic particle of cocaine from a fiber, collecting the analyte solution in a nanospray tip, and transferring the tip directly to the mass spectrometer to complete analysis. The utility of this technique is evident through the minimal sample preparation and short analysis time. The use of nanomanipulation coupled to nanospray ionization-mass spectrometry could improve on current trace particulate analysis by reducing both detection limits and sample size required to complete analysis.