Measurements of the charge and size of heptane droplets generated by electrostatic sprays showed that the droplet charge-to-volume ratio is a monotonically decreasing function of size. In the useful range of electrospray operation, characterized by droplets smaller than the size of the orifice from which the liquid is issued, it was found that the larger were the droplets the closer they were to the Rayleigh limit. In particular, when droplets had charging levels between 70% and 80% of such limit, they were observed to rupture because the repulsive force due to surface charge evidently overcame surface tension. The rupture phenomenon, here termed Coulomb fission, was also captured in microphotographs that typically showed a droplet with one or two, diametrically opposed, conical protrusions terminating in a fine jet ejecting a stream of much smaller, apparently equisized offsprings. The process appeared swift and, yet, well ordered, quite different from the common view of a violent, convulsive explosion. Corroborating evidence on the disruption pattern was also gathered by quantitative measurements of the evolution of the droplet size distribution in evaporating sprays using phase Doppler anemometry (PDA). Implications of these findings are finally discussed in the context of a particular application of electrostatic sprays, electrospray ionization, a technique that is revolutionizing the mass-spectrometric analysis of large biomolecules.

The utility of ion mobility spectrometry (IMS) for separation of mixtures and structural characterization of ions has been demonstrated extensively, including in biological and nanoscience contexts. A major attraction of IMS is its speed, several orders of magnitude greater than that of condensed-phase separations. Nonetheless, IMS combined with mass spectrometry (MS) has remained a niche technique, substantially because of limited sensitivity resulting from ion losses at the IMS-MS junction. We have developed a new electrospray ionization (ESI)-IMS-QTOF MS instrument that incorporates electrodynamic ion funnels at both front ESI-IMS and rear IMS-QTOF interfaces. The front funnel is of the novel "hourglass" design that efficiently accumulates ions and pulses them into the IMS drift tube. Even for drift tubes of 2-m length, ion transmission through IMS and on to QTOF is essentially lossless across the range of ion masses relevant to most applications. The rf ion focusing at the IMS terminus does not degrade IMS resolving power, which exceeds 100 (for singly charged ions) and is close to the theoretical limit. The overall sensitivity of the present ESI-IMS-MS system is comparable to that of commercial ESI-MS, which should make IMS-MS suitable for analyses of complex mixtures with ultrahigh sensitivity and exceptional throughput.

Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.biomarker verification | systems biology | targeted proteomics S elected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), has recently emerged as a promising technology (1-16) for high-throughput mass spectrometry (MS)-based quantification of targeted proteins in biological and clinical specimens (e.g., tumor tissues). SRM has demonstrated relatively good selectivity, reproducibility (or precision), and sensitivity for a range of multiplexed protein assays (2, 4-8, 11, 17, 18) and has potential for quantifying protein isoforms (19) and posttranslational modifications (PTMs) (3,(20)(21)(22) for which good quality antibodies often do not exist. Nevertheless, a major limitation of current SRM technology is insufficient sensitivity for detecting low-abundance proteins present at sub-nanogram per milliliter levels in human blood plasma or serum and extremely low-abundance proteins in cells or tissues. Without sample prefractionation, liquid chromatography (LC)-SRM measurements have been limited to only moderately abundant proteins in human plasma present in the low microgram per milliliter range (2,6,8).More recently, the combination of immunoaffinity depletion and fractionation by strong cation exchange (SCX) chromatography (4, 23), along with advances in MS sensitivity (24), has extended SRM quantification of plasma proteins to low nanogram per milliliter levels (4, 23). SISCAPA (stable isotope standards and capture by anti-peptide antibodies) coupled with SRM demonstrated quantification of target proteins in the same range, using as little as 10 μL of human plasma (8,(25)(26)(27). SISCAPA assays have some distinct advantages over conventiona...

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.