Recent Advances in Mass Spectrometry

Table 1. A summary of selected emerging data-independent analysis/HSM approaches and their respective pros and cons.
Method	Manufacturer	Instrument type	Brief description	MS1 isolation window	Duty cycle, m/z range	Advantages	Disadvantages	Potential impacts on proteomics
MS^E	Waters	Q-TOF	The collision cell switches rapidly and continuously between low and elevated collision energy states. At low energy state, no fragmentation occurs and precursor ion spectra are recorded. At high energy state, ramped collision energy is used to generate fragment ions. Product ions spectra of all precursors are recorded. Complex sample requires efficient separation using UPLC or further with ion-mobility separation.	Typically, 300–2000 Th (filter low mass ions)	1 s (0.5 s for intact/precursor ions scan and 0.5 s for fragment ions scan), m/z 300–1990	Requires a minimum method development. Need no prior knowledge of the sample components. Unbiased data acquisition, regardless of the ion intensity. Closes to 100% duty cycle and potentially allow a higher number of proteins to be identified. Label-free quantitation.	The method is not a replacement of DDA and is limited by the extent to which correct assignation of fragment ions to precursors is achieved. Identity of the peptides may require further validation with directed DDA. US FDA determination can only achieved with third party package.	Circumvent the issues surrounding the stochastic sampling of precursors in DDA approach. Significantly enhance sequence coverage and hold promises to locate the sites of post-translation modifications.
AIF	Thermo scientific	Benchtop Orbitrap and quadrupole-Orbitrap	The system is set to perform alternating MS scans of the precursor ions and AIF scans in which all the precursors are transferred to the HCD cell and fragmented.	None	2 s (1 s for intact/precursor ions scan and 1 s for fragment ions scan), m/z 100 –1600	Same as MS^E but with wider dynamic range and higher mass accuracy due to the use of Orbitrap mass analyzer. MaxQuant allows FDR estimation.	MaxQuant's peak recognition algorithm is not as sophisticated as IDENTITY.	Same as above
SWATH-MS	AB Sciex	Q-ToF	The system performs MS2 scan in a sequential 25 Da MS1 windows. Fragmented ions are measured with high resolution TOF mass analyzer. During data post-processing, SWATH-MS spectra are extracted to SRM chromatographs.	25 Th	3.2 s, m/z 400 –1200	Allow quantitative assay of complex samples in the same manner as SRM but enjoy the potential to monitor a higher number SRM transitions than conventional SRM and able to refine the SRM transitions retrospectively.	Incompatible with database search, and thus does not confirm the identity of the peptide detected. Require validation.	Fill the gap between discovery-based and hypothesis driven investigation. To test new hypotheses without the need to repeat the entire experiment and expedite biological discovery. Further targeted or routine analysis can be performed by SRM or MRM^HR
MSX	Thermo scientific	Benchtop quadrupole-Orbitrap	An inclusion list containing a sequence of ~5000 isolation windows is defined in Skyline before an experiment. During an analysis, the instrument loops through the inclusion list sequentially, choosing five isolation windows for each MSX scan. When an MSX scan is acquired, each isolation window is isolated, fragmented and trapped serially in time before mass analysis in the Orbitrap. The MSX spectra are demultiplexed during data post-processing and are extracted to SRM chromatographs.	5 × 4 Th	3.5 s (with 2 MS1 scans), m/z 500-–900; max. fill time for each isolation window 20 ms, Orbitrap resolving power: 17,500	Same as above An improved DIA/SWATH-MS method that has a reduced chemical background and fragment-ion interference in the extracted SRM chromatography bought by increasing the precursor selectivity. MS1 scan can be acquired at the same experiment and this produces complementary quantitative data.	Same as above The method is a compromise between the data quality and the scanning rate of system.	Same as above. The method potentially improves the precision and accuracy of the measurement relative to the SWATH-MS.
PRM	Thermo Scientific	Benchtop quadrupole-Orbitrap	A quadrupole mass filter isolated narrow mass windows. Fragmented ions are measured with ultra-high resolution Orbitrap mass analyzer.	2 Th	Maximum fill times of 3s, at resolution of 70 000 (at m/z 200), and target AGC values of 3e6	Increase the number of peptides to be monitored in one experiment. Taking the advantages of the high-resolution and accurate mass measurement to increase the degree of selectivity such that the targeted peptides can be discriminated against background interferences. High selectivity, dynamic range, and sensitivity. The MS2 spectra are compatible with database search.	The number of precursor ions that can be monitored is dependent on the duty cycle or transient length of the mass analyzer. Although a higher number of precursor ions can be monitored than SRM, the number precursor ions are still limited in an experiment. The same argument applies to pSRM/MRM^HR	Advancing targeted proteomics.

AIF: All-ion fragmentation; DDA: Data-dependent acquisition; DIA: Data-independent analysis; HCD: Higher energy collisional dissociation; IDENTITY: Ion Accounting algorithm; MRM: Multiple reaction monitoring; MS: Mass spectroscopy; PRM: Parallel reaction monitoring; pSRM: Pseudo-SRM; Q-TOF; Quadrupole orthogonal acceleration time-of-flight; UPLC: Ultra-performance liquid chromatography.

Box 1. Emerging SRM-associated MS techniques.
DMS/FAIMS is a technique separating gas-phase ions based on the difference between ion mobility in high and low electric fields at or near atmospheric pressure. By adjusting the separation voltages and the compensation voltage, the trajectory of a particular ion can be modulated. Interfering ions are set to collide with one of the electrodes (and neutralized) whereas the targeted ions migrate toward the orifice of the DMS device and enter the mass spectrometer [91]. The technique acts effectively as an ion filter thereby enhancing signal-to-noise. MRM³increases the specificity of SRM detection by using resonant excitation to fragment primary product ions trapped in the Q3 linear ion trap. The detector eventually scans the secondary product ions produced. A two-stage SRM transition is used to reconstruct an MRM³ion chromatogram [31] and therefore the method has a high specificity.

MRM: Multiple reaction monitoring; SRM: Selected reaction monitoring.

Sidebar

Key Issues

Data dependent acquisition employed in shotgun proteomics and selected reaction monitoring (SRM) employed in targeted proteomics have a number of short-comings that limited researchers from examining complex biological systems in terms of depth and width.
Emerging mass spectrometry approaches, known as data independent analysis and hyper reaction monitoring, riding on the advantages of high speed and high-resolution hybrid mass spectrometers (e.g., Q-ToF and Orbitrap), could address the short-comings of DDA and SRM.
One such approach eliminates the ion selection stage and produces alternating scan of precursor ions and fragments of all the precursors. The resulting product ion spectra are highly convoluted, but an algorithm capable of correlating the products with the precursors based on the retention time, chromatographic peak shapes, charge state, etc., for the ions is available. Enhancements can be brought by the use of ultra-performance liquid chromatography and ion-mobility gas-phase separation.
An alternative approach sequentially fragments all the ions in a relative wide isolation window, typically from 10–100 Th. The resulting product ion spectra are convoluted but can be searched against theoretical reference spectra or be mined with SRM-like targeted data extraction.
There are also novel targeted methods that sequence the precursor ions in a relative narrow precursor isolation window in a way similar to SRM. However, these methods do not measure the SRM transitions but to record the whole product ion spectra. The spectra can either be searched against conventional database or extracted with SRM-like ion chromatograph.
There is also a strategy, in which multiple but separated narrow precursor isolation windows are analyzed simultaneously. The multiplexed spectra are demultiplexed and extracted to SRM ion chromatographs. Such approach enjoys the sampling rate of the methods using wide isolation windows but has the selectivity of the methods using relative narrow precursor isolation windows.
These new approaches are not faultless themselves and are not substitution to data dependent acquisition or SRM. However, they are useful additional tools or new opportunity for biomedical and bimolecular investigations and may produce an impact to the field in the next few years.