In recent years, the combination of liquid chromatography and high-resolution exact mass tandem mass spectrometry has been widely adopted for a variety of research applications such as drug discovery and development; proteomics; and, more recently, biomarker discovery. From an analytical perspective, both the profiling and characterization of the typical samples encountered in this arena can be extremely challenging since they often contain thousands of components over a wide range of concentrations, and individual compounds of interest can carry numerous or complex modifications.
High-resolution mass spectrometric approaches (based on time-of-flight [TOF], Fourier transform [FT], or high resolution ion traps) have been employed extensively to further increase the quantitative and qualitative information obtained in such applications. However, it is becoming increasingly important for the base performance characteristics of the MS system to be routinely accessible at high acquisition rates in order to take full advantage of separation technologies such as ultrahigh-performance liquid chromatography (sec), gas chromatography (sec), and ion mobility separations (msec). Unlike FT-MS or high-resolution ion-trap instrumentation, the geometry and fast experimental times afforded by the high-resolution TOF-based platform described here provide very high levels of exact mass MS and MS-MS performance for quantitative or qualitative analysis, and new enabling analytical possibilities through the introduction of enhanced, integrated ion mobility technology.
System description
Figure 1 - Schematic of SYNAPT G2 system. Triwave™ is the enabling next-generation IM technology and consists of three T-Wave devices. The TRAP T-Wave traps and accumulates ions, after which the ions are released in a packet into the IMS T-Wave, where ion mobility separation is performed. The TRANSFER T-Wave delivers the mobility separated ions to the oa-TOF mass analyzer. Ion arrival time (or drift time) distributions are recorded by synchronizing the oa-TOF mass spectral acquisitions with the gated release of ions from the TRAP T-Wave device. To provide increased ion mobility resolution, higher N2 pressures and higher T-Wave pulse amplitudes were required compared to those on a first-generation SYNAPT instrument. A high-pressure helium-filled cell has been introduced at the front of the IMS T-Wave cell to minimize scattering and/or fragmentation as ions are introduced into the high N2 pressure region. QuanTof™ is the enabling next-generation TOF analyzer of SYNAPT G2. QuanTof’s high field pusher and dual-stage reflectron, incorporating high-transmission parallel wire grids, reduce ion turnaround times due to prepush kinetic energy spread and improve focusing of high-energy ions, respectively. The ion detection system combines an ultrafast electron multiplier and hybrid analog-to-digital conversion (ADC) detector electronics to provide high resolution and low noise at low ion currents, high quantitative performance and exact mass at high ion currents, all at the elevated data acquisition rates of ion mobility analyses. The hybrid ADC system comprises an 8-bit ADC sampling at 3 GHz feeding to a field-programmable gate array (FPGA) for signal processing, and subsequently to a block of memory for accumulating the 200 sequential mass spectra that form the mobility arrival time spectrum.
The SYNAPT™ G2 (Waters Corp., Milford, MA) hybrid quadrupole/ion mobility (IM)/orthogonal acceleration-time of flight (oa-TOF) geometry is shown in Figure 1. Ions are generated using an atmospheric pressure ionization (API) source, passed through a quadrupole mass filter to the IM section of the instrument (Triwave), containing three traveling wave (T-Wave) ion guides, and then into the high-resolution quantitative TOF analyzer (QuanTof) for mass analysis. (Note: the traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,206,506; 1993.)
The system provides two fundamental modes of operation: 1) TOF mode for high-resolution, exact mass MS, and MS-MS performance, and 2) High Definition MS™ (HDMS) mode to combine TOF mass spectrometry with high-efficiency ion mobility separations (Figure 1). The QuanTof is a next-generation oa-TOF platform (Figure 1) that provides high resolution (up to 40,000 FWHM), exact mass (<1 ppm root mean square [RMS]), increased sensitivity, and quantitative performance over 5 orders of linearity, all at high acquisition rates (up to 20 spectra/sec).
The system incorporates T-wave ion mobility separation technology within a dual-collision cell arrangement (Triwave) providing increased IM resolution (up to 4× higher than the first-generation SYNAPT) similar to the latest purpose-built drift tube IM-MS research systems.
High performance at high acquisition rates
Dynamic range and sensitivity
The LC-MS analysis of complex biological samples can often give rise to very complex spectra at any time period over the course of a chromatographic run and can contain hundreds or even thousands of peaks (including different isotopes and charge states) over a wide range of intensities. It is therefore critical that the mass analyzer has the capacity to separate and detect hundreds of ion species at any given time with high mass accuracy across a wide range of concentrations.
Figure 2 - In-spectral dynamic range of over four orders of magnitude for 1-sec electrospray ionization (ESI)-MS analysis of bovine insulin. The [M+5H]5+ ion at 1147.5 m/z and background ion at 364 m/z (inset) can be visualized simultaneously due to the ability of the high charge capacity of the TOF analyzer (an estimated 14 million charges are in the spectrum displayed here).
As shown in Figure 2, the SYNAPT G2 system provides an in-spectrum dynamic range of over four orders of magnitude. Under all practical situations, the TOF analyzer does not suffer from space charge effects (unlike a high-resolution ion trap, which has a capacity of approximately 2× 106 charges)1; thus there is no compromise in dynamic range, detection limits, or TOF resolution as the complexity of the analyte increases. This is, of course, critical for the unambiguous detection and identification of low-level components, which can often be the analytes of the most interest.
Figure 3 - Quantitative data obtained from the nanoscale UPLC® (Waters Corp.)-ESI-MS analysis of the epidermal growth factor receptor (EGFR) phosphopeptide (GSTAENAEYpLR) at m/z 645.8 (2+) spiked into a constant background of 100 fmol of bovine serum albumin (BSA) demonstrating phosphopeptide linearity over at least four orders of dynamic range with exact mass measurement.
Furthermore, quantitative measurements can be made over a wide dynamic range with the confidence of exact mass (for confirmation of identity). This is demonstrated in Figure 3 for the quantification of a known peptide in a peptide mixture over four orders of magnitude.
High resolution, mass measurement accuracy, and precision
Figure 4 - UPLC/MSE fragment ion spectrum of Peptide T9 (FTISADTSK) from a humanized monoclonal antibody acquired at 10 spectra/sec with >30,000 FWHM mass spectral resolution. Data were acquired from a UPLC peak width of 4 sec at half height >30,000 FWHM. A 1.5-mDa window was used to generate extracted ion chromatograms (inset) for the precursor and fragment ions.
Figure 4 demonstrates the ability of oa-TOF to deliver high resolution at the fast spectral acquisition rates required to keep pace with ACQUITY® UPLC (Waters Corp.) separations, which typically deliver peak widths as low as 1 or 2 sec at half height. With a spectral acquisition rate of up to 20 spectra/sec, the oa-TOF system ensures that sufficient points can be obtained to generate accurate LC peak profiles with high mass resolution to maximize the ability to resolve compounds and provide exact mass measurement. The ability of the oa-TOF system to deliver high mass accuracy and accurate isotope ratios significantly aids in the identification of small molecules through the elimination of false positives.