Off to a good start with the ion source
As the first component of an MS system, the ion source is important because it’s responsible for ionizing as many of your sample peptides as possible. “If you’re not producing and capturing a lot of ions, then you’re inherently limited in sensitivity,” says Christine Miller, ‘omics market manager at Agilent Technologies. “The initial step is probably the key step, which is how do you make ions, and then how do you sample them.”
Two aspects of Agilent’s iFunnel ion-source technology enable proteomics researchers to obtain better ion generation. One is the company’s proprietary Jet Stream technology, in which the electrospray created during ionization is ensheathed by a gas, which serves both to dry and to collimate—or collect and focus—the ions. The advantage is in directing “a tighter packet of ions right in front of the mass spectrometer entrance,” says Miller, “which means that we’re then able to sample a larger percentage [of ions].” Another aspect is a dual-stage ion funnel that removes gas and focuses ions into the mass analyzer.
One of Agilent’s newest MS system, the 6495 Triple Quadrupole, uses iFunnel technology. Though ion funnels have been available for several years, not all MS systems have them, and not all MS vendors offer them. Ion funnels are beneficial because they help capture ions, which improves sensitivity. According to Miller, the ion funnel and Jet Stream technology boost sensitivity by 5- to 10-fold and by 3- to 5-fold, respectively, compared with using conventional electrospray ionization.
Another ion-source development is Waters’ StepWave technology, which increases sensitivity, according to Hans Vissers, Waters’ senior manager of science operations in health sciences research. Built like a stack of rings, the StepWave works by enhancing ion collection and transmission from the ion source to the entrance of the mass analyzer while also removing non-ionized, neutral contaminants.
Waters recently introduced the Rapid Evaporative Ionization Mass Spectrometry™ (REIMS™) Research System with iKnife™ Sampling, a method of ionizing nontraditional samples under ambient conditions that wouldn’t be appropriate for normal sample preparation or chromatographic separation protocols. REIMS directly heats a sample, which forms an aerosol that is sampled by the system. Sample identification is possible because the vapor contains specific chemicals that the system analyzes by time-of-flight (TOF) MS. This system can be used to determine the differences in sample tissues in application areas as diverse as food testing and species identification, as well as reaching ways to potentially provide surgeons with diagnostic information about the tissue they are cutting, in real time.
Nanospray, another ion-generating process, is useful for researchers using small sample volumes, as in the case of precious or rare sample material. Bruker Daltonics provides an ion-source improvement with its CaptiveSpray nanoBooster, “a nanospray source that increases reproducibility and makes ionization more versatile, because you can ionize a wider range of compounds,” says Pierre-Olivier Schmit, proteomics market area manager at Bruker Daltonics. Bruker’s CaptiveSpray technology uses a vortex gas that surrounds and focuses the ionized peptides emerging from the electrospray. It helps make nanoflow liquid chromatography (LC)-MS systems—whose small diameters are more prone to clogging—more stable, robust and capable of reproducible results. The nanoBooster, which does enrich the carrier gas with organic solvent vapor, can increase the ionization efficiency for a wide range of compounds.
Through the mass analyzer
After ionization, the sample particles enter the mass analyzer, which separates them based on their charge-to-mass ratio. Mass analyzers typically found in systems used by proteomics researchers include the TOF, quadruple, ion trap and orbitrap varieties.
The ion-beam compressor technology in Agilent’s TOF and qTOF analyzers helps concentrate ions for better transmission to the detector. Agilent’s 6495 Triple Quadrupole system also features advances in its curved collision cell, which enhances sensitivity by reduction of chemical background. Similarly, Waters’ hybrid TOF mass analyzers use XS collision cell technology that reduces the dispersion of ions as the ion beam arrives at the mass analyzer.
Thermo Fisher Scientific recently updated both its quadrupole and ion trap analyzers in the Orbitrap Fusion Lumos tribrid mass spectrometer. The quadrupole analyzer is essentially a series of mass filters that allows isolation of compounds with improved efficiency. Historically, it has been difficult to select a narrow mass range without impeding ion transmission, because fewer ions make it to the detector. But Thermo’s mass filters accommodate mass windows down to 0.4 atomic mass unit (amu) while maintaining high transmission, according to Andreas Huhmer, director of marketing in proteomics at Thermo Fisher Scientific.
Thermo uses its linear ion trap analyzer to make electron transfer dissociation (ETD) more efficient. During ETD, cationic peptides react with an anionic reagent, producing fragmentation that enables researchers to analyze the peptide backbone. Thermo recently released its Orbitrap Fusion Lumos Tribrid Mass Spectrometer. This system is especially useful for analyzing intact proteins using fragmentation techniques. “With this particular instrument, the fact that you can combine high-energy collisional dissociation (HCD), collisional induced dissociation (CID), as well as this improved ETD, in combination with the high-speed orbitrap, you can get a lot of information,” says Huhmer.
Bruker Daltonics has made advances in the analyzer of its new impact II™ and maXis II™ system, an ultra-high-resolution quadrupole TOF instrument, for improved mass accuracy and resolution. The maXis II is particularly useful for applications such as characterizing heavy and light chains of monoclonal antibodies, including their deamidated variants, for example. The system’s High Mass Option is also suitable for analyzing protein complexes and antibody drug conjugates.
Ending with the detector
After traversing the mass analyzer, ions are transmitted to the detector and converted into a digital signal. Ionized peptides emerge from the analyzer and hit the detector plate, whereupon a cascade of electrons is emitted (usually via electron multipliers or microchannel assemblies)—and the signal is digitized. For TOF-based instruments, this ion-conversion step can be improved by making it faster (i.e. shorter latency) which improves resolution, says Miller. Agilent has accomplished this on its 6550 and 6545 Q-TOF systems. On the Agilent 6495 Q-TOF system, improved high-energy conversion dynode technology has increased ion conversion by about 2- to 3-fold.
Instrument companies also are improving detectors, by widening dynamic range. “As proteomics [moves] to more targeted and more quantitative applications, dynamic range becomes an important element,” says Miller. Waters offers a new, dual-channel QuanToF2 detection system for its TOF MS systems, which adds an additional order of magnitude of dynamic range compared with previous-generation detection systems, says Vissers, and it is included in the new Vion IMS QToF system. “A challenge for mass spectrometry based quantitative methods compared to othertechniques, for example clinical assays, is its dynamic range in relation to the dynamic range of a biological sample,” says Vissers. “But it is expected that this issue, in combination [with] on-line sample preparation and separation techniques, can be addressed in the near future. Sciex has adjusted the detector in its Triple TOF 6600 system to improve dynamic range to up to five orders of linear dynamic range for SWATH analysis of complex proteomics samples, according to Aaron Hudson, senior director of academic and clinical research business at Sciex.
As instrumentation continues to improve, the integration of MS in more common experimental workflows is occurring. “Antibodies are the de facto standard for measuring low-abundance proteins, but antibodies sometimes detect the wrong things, and their quality varies,” says Hudson. Today, for example, he sees researchers begin with immunocapture of a specific peptide, followed by MS on the captured species using multiple reaction monitoring (MRM) based triple quadrupole systems. “That combination of the sensitivity and pull-down [provided by the antibody], along with the specificity of mass spectrometry, is going to be really important in the next few years,” says Hudson.
MS analysis of protein complexes is another trend that’s beginning to make inroads in a previously daunting area. The challenges are many, says Huhmer, beginning with making a complex that will even enter the instrument. Manipulating complexes within the mass spectrometer without having them immediately fall apart, or stick together too strongly, is another challenge. But he is optimistic that continual advances in MS technology—especially in highest-end instruments—will enable researchers to solve these problems.
From robust workhorses for the nonexpert to high-end instruments for studying protein complexes, MS systems continue to improve as MS tool providers evolve the technology to support proteomics researchers. Whatever your area of proteomics, there is likely an MS system that will open new doors in your proteomic studies and discoveries.