Improving Liquid Chromatography-Mass Spectrometry Sensitivity Using a Subambient Pressure Ionization with Nanoelectrospray (SPIN) Interface
In this work, the subambient pressure ionization with nanoelectrospray (SPIN) ion source and interface, which operates at ~15–30 Torr, is demonstrated to be compatible with gradient reversed-phase liquid chromatography-MS applications, exemplified here with the analysis of complex samples (a protein tryptic digest and a whole cell lysate). A low liquid chromatographic flow rate (100–400 nL/min) allowed stable electrospray to be established while avoiding electrical breakdown. Efforts to increase the operating pressure of the SPIN source relative to previously reported designs prevented solvent freezing and enhanced charged cluster/droplet desolvation. A 5- to 12-fold improvement in sensitivity relative to a conventional atmospheric pressure nanoelectrospray ionization (ESI) source was obtained for detected peptides.
Key wordsESI LC-MS Sensitivity Subambient pressure ESI Ion source
Electrospray ionization-mass spectrometry (ESI-MS) [1, 2] is widely applied to a variety of chemical and biological applications due to its broad ability to efficiently create gas-phase ions from solution, and ease of direct coupling with liquid separation techniques [3, 4, 5]. The sensitivity of ESI-MS is largely determined by a combination of the effectiveness of producing gas-phase ions from analyte molecules in solution (ionization efficiency) and the ability to transfer the charged species from atmospheric pressure into the low-pressure region of the mass analyzer (transmission efficiency) [6, 7, 8, 9]. In spite of the high ESI efficiency, especially at low nL/min flow rates [10, 11, 12, 13, 14], large ion losses can occur during transmission from atmospheric pressure to the low pressure region of the mass analyzer [6, 15, 16]. With advances in MS instrumentation, the greatest losses now typically occur at the interface region of the ESI-MS, where a small orifice or a heated capillary inlet (which, e.g., samples only a small portion of the electrospray) leads to significant losses and reduction in achievable instrument sensitivity [6, 9, 11].
An approach we have been developing for eliminating losses associated with the MS inlet involves removing the inlet entirely and placing the ESI source in a lower pressure region , where an ion funnel can transmit ions with high efficiency. Electrospraying of conductive and low volatility liquids at very low pressures was initially developed for space propulsion applications , and its implementation as an ion source for mass spectrometry was referred to as electrohydrodynamic ionization (EHD) . This source has proven to be effective with highly nonvolatile liquids at very low flow rates, such as glycerol , liquid metals [20, 21], and ionic liquids  that prevented the electrospray cone from freezing or boiling by lowering the evaporation rate and allowing sufficient heat transfer at lower pressure. However, these nonvolatile liquids are incompatible with typical liquid-phase separation techniques for routine MS analyses that involve the use of volatile solvents and higher flow rates. An early low-pressure ESI source concept applicable to standard liquid chromatography (LC) solvents described in a 1992 patent  involved positioning a metal-coated, tapered electrospray emitter at the entrance of a quadrupole ion guide in a <0.01 Torr chamber, but no results were reported. A later patent by a different group describes a low pressure ESI source at <0.1 Torr followed by a chamber at an elevated pressure to facilitate droplet desolvation and solvated ion declustering . This arrangement was subsequently reported to be capable of detecting 500 ng of caffeine injected onto an LC column , but suffered significant losses associated with the need to increase pressure after the ESI source chamber and inefficient declustering/desolvation due to practical limitations on the pressure in that region (i.e., ion losses increase as pressure is increased in order to make declustering/desolvation more efficient). All the ESI sources described in these patents operate electrospray at pressures of <0.1 Torr, far below the pressure range most prone to electrical breakdown (~1 Torr). Regardless, the low ESI operating pressures used in these sources degrade electrospray performance (e.g., due to boiling, freezing, and inefficient evaporation of the solvent), which precluded their practical implementation on ESI-MS.
The subambient pressure ionization with nanoelectrospray (SPIN) ion source and interface has been developed to address difficulties associated with desolvation, ion production, and transmission in a reduced pressure environment . Central to the implementation of the SPIN source is the use of an electrodynamic ion funnel  operated in the 15–30 Torr pressure range. The ion funnel is a variation of a stacked ring ion guide  created by a series of closely spaced electrodes in which a radio frequency (rf) voltage and a direct current (DC) voltage are applied on each electrode to both confine ions and push ions “drifting” towards the funnel exit. The aperture of the ion guide decreases gradually down to the inner diameter (i.d.) of the conductance-limiting orifice at the exit. The elevated SPIN source operating pressure relative to previous low pressure ESI sources avoids the electrical breakdown at the electrospray onset voltages, and the relatively long residence time in the ion funnel enhances charged cluster/droplet declustering/desolvation, allowing the resulting gas phase ions to be effectively generated and transmitted into the mass spectrometer. In an initial demonstration, the SPIN source has been shown to effectively ionize infused samples using a variety of LC-compatible solvents providing an ~5-fold increase in detection sensitivity compared to a standard heated inlet capillary ESI source and interface design . The previously reported results indicated that ion production efficiency in the SPIN source was similar to the one in atmospheric pressure ESI source, while sample loss due to ion transmission through an inlet was completely eliminated in the SPIN source design.
Although promising, these experiments used the infusion of easily electrosprayed standard solutions with 50% organic solvent (methanol or acetonitrile). Many MS analyses require a liquid-phase separation before MS detection for better dynamic range, selectivity, and sensitivity, and it is crucial to evaluate the SPIN source with a broarder range of solvent compositions, such as those commonly encountered with gradient-elution LC separations. Here, we report on coupling gradient reversed-phase LC separations with the SPIN source to separate and analyze complex proteomic samples. Chromatographic and MS peak intensities obtained using the SPIN source at 15–30 Torr and a standard atmospheric pressure ESI source with a typical heated capillary inlet are compared for several peptides. These results further support the utility of the SPIN source and point the way to ESI source and interface designs approaching ideal MS detection sensitivity.
Formic acid (FA, Sigma-Aldrich, St. Louis, MO, USA) and trifluoroacetic acid (TFA, Sigma-Aldrich) based LC mobile phases were employed in the experiments reported here. The FA-based mobile phase A consisted of 0.1% FA in purified water (Barnstead Nanopure Infinity System, Dubuque, IA, USA), and mobile phase B consisted of 0.1% FA in acetonitrile (Fisher Scientific, Pittsburgh, PA). The TFA-based mobile phase A consisted of 0.2% acetic acid (Sigma-Aldrich) and 0.05% TFA in purified water, and mobile phase B consisted of 0.1% TFA in 90% acetonitrile and 10% purified water. Tryptic digests of bovine serum albumin  (BSA; Pierce Biotechnology, Rockford, IL, USA) and Shewanella oneidensis  (grown in-house) were prepared using sequencing grade trypsin (Promega, Madison, WI, USA) according to previously reported procedures and the samples were diluted to final concentrations of 0.1 μg/μL and 0.05 μg/μL, respectively.
A Gilson (Middleton, WI, USA) 321 high-performance liquid chromatography (HPLC) system was used to provide an exponential gradient  at a flow rate of ~300 nL/min for the analysis of the BSA tryptic digest using TFA-based LC mobile phases. An Agilent 1100 Series LC pump (Santa Clara, CA, USA) was used to provide a pre-programmed gradient (minute:percentage of mobile phase B = 0:5%, 20:12%, 85:35%, 97:60%, 100:95%) at flow rates between 100–400 nL/min for the analysis of the Shewanella oneidensis samples using FA-based LC mobile phases. Reversed-phase capillary LC columns were prepared in-house by slurry packing 3-μm Jupiter C18 stationary phase (Phenomenex, Torrance, CA, USA) into a 60-cm long and 75-μm-i.d./360-μm-o.d. fused silica capillary tubing (Polymicro Technologies, Phoenix, AZ, USA). Sample loading was accomplished using Valco valves (Valco Instruments Co., Houston, TX, USA) with a 5-μL sample loop.
Electrospray emitters were fabricated by chemically etching sections of either 5- or 10-μm-i.d./150-μm-o.d. fused silica capillary tubing (Polymicro Technologies), as described previously . Hydrophobic treatment of the emitters was accomplished via plasma polymerization and deposition of C4F8 using a PlasmaLab 100 inductively coupled plasma etch and deposition system (Oxford Instruments, Oxfordshire, UK). The emitters were affixed to a silicon wafer, loaded into the plasma chamber, and exposed to 160 sccm of C4F8 for 30 s with an ICP power of 2 kW and a frequency of 13.56 MHz. Following this first deposition, the emitter was turned 180° and the procedure was repeated to ensure complete and uniform coating.
A stainless steel union was used to attach the electrospray emitter to the LC column and also served as the connection point for the electrospray voltage (Bertan 205B-03R; Hicksville, NY, USA or Ultravolt Rack X-250, Ronkonkoma, NY, USA). To produce ions at subambient pressure, the emitter was inserted into the vacuum chamber via a vacuum feedthrough as described in detail previously . The conical outlet in the previous design was replaced by a cylindrical outlet, with the emitter protruding ~2 mm beyond the cylindrical outlet into the vacuum chamber. This change allowed the emitter tip to be continuously monitored with a Hitachi KP-D20BU video camera (Toyama, Japan) equipped with a long distance microscope (Edmund Optics, Barrington, NJ, USA). In these conditions, the optimum voltage driving the electrospray was ~6000 V.
2.4 Mass Spectrometry
Mass spectra were acquired in the 300–2000 m/z range in positive ESI mode with a 0.1 m/z step size on an Agilent MSD1100 (Santa Clara, CA, USA) single quadrupole mass spectrometer equipped with a dual ion funnel interface . The standard atmospheric pressure ESI source used a 7.6 cm long, 490 μm i.d. stainless steel inlet capillary heated at 120 °C and biased 20 V higher than the first ion funnel electrode. The interface chambers hosting ion funnels were respectively pumped by EM28 and EM18 rough pumps (BOC Edwards, Wilmington, MA, USA) down to 15–30 and 1.7 Torr (measured at the pump throat) for each arrangement.
3 Results and Discussion
Electrical breakdown and wetting of the ESI emitter outer wall have previously been identified as key reasons for disruption or instability of electrosprays operated at 15–30 Torr [17, 32, 34]. Our initial studies indicated that the SPIN source could be operated without significant challenges when infusing solutions containing 50% organic solvent (methanol or acetonitrile) . Leaking heated CO2, an electron scavenger, inside the SPIN source chamber allowed operation at even lower pressures with no electrical breakdown . However, electrospray disruption due to emitter outer wall wetting and liquid beading was still a challenge for the operation over the full range of liquid compositions characteristic to gradient reversed phase LC separations.
The difficulties due to emitter wetting were found to be significantly alleviated by reducing the i.d. of the electrospray emitter from 10 to 5 μm. While the reason for improved performance is not clear, we speculate that the inertia due to increased flow velocity through the smaller emitter minimizes the interaction between the liquid and the solid surfaces. Improved performance was also achieved by coating 10 μm i.d. emitters with a hydrophobic layer, suggesting that interfacial forces at the liquid-solid interface play a significant role in electrospray disruption at decreased pressure. The experiments reported herein were performed on the SPIN source with reversed-phase LC using 5 μm i.d. emitters to enable stable electrospray performance during the entire LC reversed phase gradient.
The S/N ratios are provided on top of each bar of Figure 2. The S/N ratio is expected to increase with the square root of the signal if only shot noise is present in the experiment. An example is the increase of ~3 times of the S/N ratio for the peptide with m/z 601.4 for an increase of the signal by a factor of ~10. In other cases (e.g., the peptides with m/z 602.8 and 558.7) the S/N ratios do not improve with the same scaling, suggesting S/N is largely defined by ‘chemical noise’ levels. There are also cases (e.g., the peptides with m/z 1188.2 and 717.2) where the S/N ratio was larger using ESI, and thus suggesting that background species that were not ionized or that were suppressed by the ESI source were contributing to the noise in the SPIN experiments. Certain types of measurements cannot take advantage of an increase in signal unaccompanied by a corresponding increase in the S/N ratio. However, measurements able to exclude or reduce the chemical noise or other interferences (for example MRM measurements) may take full advantage of the larger ion population provided by the SPIN source.
The present results showing higher signal intensities at lower LC flow rates may seem to contradict the previously published results , which indicate lower intensities at decreasing infusion flow rates. However, we need to point out that the flux of analyte delivered to the source in an infusion experiment decreases with the flow rate, which may not always be the case when using a LC separation. Since no significant changes in the widths of the chromatographic peaks were observed in the flow rate range explored by our experiments, the flux of analyte delivered to the ion source was similar under the same column loading conditions. The interplay between the flow rate and the ionization efficiency in the SPIN source was discussed in detail in our previous work .
Ten Randomly Selected LC-MS Peptide Peaks from the Analysis of 0.5 μg BSA Tryptic Digest Sample with a Standard Atmospheric ESI Source and the SPIN Source and Interface
Conventional ESI source
% st. dev.
% st. dev.
The recently developed SPIN source and interface has been found to efficiently create ions in a 15–30 Torr region of a mass spectrometer and provide effective coupling of LC with MS. The initial evaluation used tryptic digest samples for reversed-phase gradient LC demonstrated that the SPIN source allowed stable nanoelectrospray operation throughout the entire reversed-phase gradient and increased the average instrument sensitivity by a factor of 5–12. The use of an ion funnel in the SPIN source provided effective droplet desolvation and enabled mass spectra similar to a conventional nanoESI source. A lower LC liquid effluent flow rate at an operating pressure of 15–30 Torr improved charged particle desolvation and declustering , as smaller droplets facilitated more efficient ion formation. In addition, this pressure regime eliminated solvent freezing, a challenge with previous low pressure ESI sources operated at <0.1 Torr . Thus, the SPIN source can eliminate the major ion losses (shown to be 80%–90% ) due to the use of a heated capillary inlet or a small orifice in the conventional ESI-MS instruments and may lead to an ESI-MS platform with optimum ion transmission efficiency from the ion source to the MS detector. Our results also suggest that significant further improvements in sensitivity may be achievable by the use of lower flow rates and conditions that further improve droplet desolvation.
Portions of this research were supported by the NIH National Center for Research Resources (RR018522), the NIH National Cancer Institute (R21 CA126191), and the National Institute of Allergy and Infectious Diseases NIH/DHHS through interagency agreement Y1-AI-4894-01. Experimental portions of this research were performed in the Environmental Molecular Sciences Laboratory, a U.S. Department of Energy (DOE) national scientific user facility located at the Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multiprogram National Laboratory operated by Battelle for the DOE under contract no. DE-AC05-76RLO 1830.
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