Advances in ionisation techniques for mass spectrometry‐based omics research

Omics analysis by mass spectrometry (MS) is a vast field, with proteomics, metabolomics and lipidomics dominating recent research by exploiting biological MS ionisation techniques. Traditional MS ionisation techniques such as electrospray ionisation have limitations in analyte‐specific sensitivity, modes of sampling and throughput, leading to many researchers investigating new ionisation methods for omics research. In this review, we examine the current landscape of these new ionisation techniques, divided into the three groups of (electro)spray‐based, laser‐based and other miscellaneous ionisation techniques. Due to the wide range of new developments, this review can only provide a starting point for further reading on each ionisation technique, as each have unique benefits, often for specialised applications, which promise beneficial results for different areas in the omics world.

with ESI being a reliable soft ionisation technique that can interface under atmospheric pressure (AP) with a range of mass analysers. In recent years however, areas in which ESI is lacking have become more apparent. While ESI is typically robust and sensitive, it has a relatively low throughput due to the common use of relatively slow LC separation prior to ionisation [5]. ESI also has significant limitations when it comes to sample preparation. Considering nano-ESI in particular (with its smaller capillary internal diameter), sample matrices are heavily limited in their final composition as desalting steps are needed prior to the ionisation stage to avoid both salt precipitation on the ESI needle and ion suppression, further reducing throughput and potentially sensitivity due to suboptimal analyte recovery [6][7][8][9]. Additionally, ESI alone cannot perform MS imaging (aside from a few niche instrumental setups [10,11]) and therefore cannot perform any omics analyses that require localisation without lengthy sample preparation procedures.
Due to these limitations, there has been a significant amount of research in recent years with the aim of improving upfront sample preparation using faster and more sensitive separation techniques F I G U R E 1 Classification of the ionisation techniques described within this review, according to spray-based, laser-based and other techniques. Note that the spray-based set consists of techniques the literature has discussed as being driven at least in part by a process similar to electrospray ionisation. While there are many more ionisation techniques available, the techniques mentioned in this review have significantly contributed to recent advances in the field of omics.
As the main ionisation technique in omics research, ESI has remained relatively static in terms of new developments in recent years. Most of the changes and associated targeted improvements in specific areas of ESI are with relation to sample additives and emitter shape [31][32][33]. While these advancements improved the analytical capabilities of ESI towards a wider variety of samples and analytes, they are not fundamentally new advancements of the technique itself.
MALDI has fundamentally made some greater advances such as liquid AP-MALDI (LAP-MALDI) as discussed below and in applications such as biotyping, imaging and high-throughput screening (HTS) in clinical and industrial application areas. In addition, there is a constant flow of developing new MALDI matrices [34,35]. As with ESI (as discussed later in this review) there have also been many new ionisation methods, modifications and hybrid techniques that use MALDI (and/or ESI) as a starting point.
In this review, we will be assessing the current landscape of newly developed ionisation techniques, first looking at (electro)spray-based, then laser-based and finally other ionisation techniques in the context of omics research. LC-MS-based proteomics, allowing for higher yields of multiply protonated ion species and therefore higher resolutions [40]. Different supercharging reagents can also be used as a mixture to target and enhance specific charge states of target peptides, as demonstrated by

SPRAY-BASED IONISATION TECHNIQUES
Van Wanseele et al. [41]. Overall, there is a wealth of applications and F I G U R E 2 General schematic for paper spray ionisation. A paper triangle is wetted with the sample solution and connected to a high-voltage power supply. This causes a plume of charged droplets to be ejected from the tip of the paper triangle towards the mass spectrometer inlet. Adapted with permission from ref. [43]. Copyright 2010 American Chemical Society a range of supercharging reagents, which have been recently reviewed by Abaye et al. [36].

Paper spray
Paper spray ionisation is a low-cost, easily accessible technique, first developed by Wang et al. in 2010 [42]. As shown in Figure 2, an aliquot of liquid sample solution (or an adequate solvent, if the analyte is already on the paper) gets deposited onto a small paper triangle, which is then placed in front of the mass spectrometer inlet. An electric field is applied to the paper, initiating ionisation of the liquid sample from the paper's tip facing the mass spectrometer's inlet. Paper spray allows for quick and facile access to a variety of sampling methodologies inaccessible to many other ionisation techniques, such as the direct analysis of dried blood spots and separated compounds from a TLC strip, and surface analysis (e.g., by wiping a surface with the paper substrate) [42,43].
It lends itself perfectly to metabolomic, lipidomic and proteomic analysis in clinical settings as biofluids can be deposited directly onto the paper substrate and then sent for analysis, increasing sample throughput (due to little sample preparation) and therefore diagnostic speed significantly [43].
While a range of applications were discussed in the original paper spray article, they were limited in scope because of significant ion suppression effects due to the substrates and elution solvents, resulting in low sensitivity [43,44]. Advances in paper spray have therefore been primarily focused on improving sensitivity, while also aiming to expand the types of analytes accessible to the technique.
The (paper) substrate material has been a major focus for increasing sensitivity. Paper spray is not a 'one-type-fits-all' technique; different analyte classes have varying affinities for and interactions with different substrates [45]. While they cannot necessarily be called paper substrates, polymeric and non-porous materials have been used as substrates in paper spray. Recently, Teslin has been used as a substrate with the aim of rapidly detecting COVID-19 metabolomic biomarkers [46]. In theory, the silinol groups in the Teslin substrate allow for greater interaction of the analytes with the charged substrate, allowing for a greater number of molecules to be ionised compared to the typical cellulose paper substrates [47]. A Teflon substrate with a conducting wire through the centre has been used to examine metabolites present in urine and saliva, with an S/N increase of at least 100-fold compared to cellulose paper [48]. Similarly, a poly(methyl methacrylate) substrate spiked with carbon nanotubes was used to analyse analyte types ranging from small molecules to mid-sized proteins with an ion signal intensity increase of 20-100 times that of cellulose paper [49].
Desalting paper spray (DPS) is another modification to paper spray that has been developed recently. By washing the substrate with an acetonitrile/water mixture prior to the analysis excess salts can be removed from the substrate. While initially utilised to reduce the analyte ion signal suppression observed in glycan and oligosaccharide analysis due to high levels of adduct ion formation [50,51], this technique has been expanded into the realm of proteomics by using a polymer substrate coated with Nafion, a fluoropolymer. The proteomic profiles of saliva samples were examined, and the Nafioncoated substrates showed a desalting efficiency of up to 90% higher when compared to control substrates [52].

Desorption electrospray ionisation (DESI)
Initially developed as an ambient ionisation technique for direct in vivo sampling and crude MS imaging (MSI) analysis in 2004 by Cooks and coworkers [53], the non-destructive nature of DESI was shown to offer distinct advantages within the field of MSI that MALDI (the leading MSI tool) could not match [54]. The primary advantage of DESI is that it does not necessarily require pre-treatment of a sample. A range of histologically compatible solvent systems are available that can be sprayed directly onto tissue samples for DESI MSI analysis, while MALDI MSI requires any tissue sample to be pre-treated with the MALDI matrix for effective ionisation to occur.
Since its inception, updates to the design of DESI sources and their coupling to MS instrumentation have been the key to unlocking access to higher spatial resolution, higher specificity and a wider range of applications. One area of these design updates lies within the design of the DESI emitter. Towers et al. modified a commercial DESI source by rerouting a high-voltage power supply through the solvent flow to create charged primary droplets, and by introducing a heated inlet capillary to aid desolvation of the secondary droplets [55]. With this setup alongside ion mobility spectrometry, it was possible to observe intact peptide and protein ion signals with a pixel resolution of 150 µm [55], an endeavour that had previously been extremely difficult outside of highly optimised conditions.
A modification to DESI was made by He et al. in 2011 [56], later called airflow-assisted DESI (AFADESI) [57]. It was introduced with the aim of achieving higher analytical sensitivities for remote DESIlike sampling by providing additional air flow to the mass spectrometer inlet, using a vacuum pump connected to a transport tube for efficient extraction of the DESI-produced sample material [56]. While initially shown to be useful in pharmaceutical and explosive residue analysis [56], AFADESI has also seen a number of novel uses in the field of omics. providing the ability to differentiate between unsaturated lipid isomers [58]. Abliz and coworkers combined AFADESI with a reagent-spiked hydrogel derivatisation methodology, allowing for a large number of metabolites and lipids to be identified from a rat brain [59].
Another DESI modification that has seen an increase in uptake recently is nano-DESI [60,61]. While DESI utilises a solvent spray at a distance from the substrate, nano-DESI employs a solvent bridge at the sample target between a primary capillary and a secondary nanospray capillary, resulting in a hybrid between liquid extraction surface analysis (LESA) and DESI [60,62]. The differences between DESI and nano-DESI are displayed in more detail in Figure 3. Modifications to this technique have allowed for great increases in versatility.
Cooper and coworkers utilised non-denaturing solvents alongside a shortened inlet capillary to gain high-resolution native proteomic profile images of intact proteins from kidney tissues [62] and brain tissues [63].
Lanekoff and coworkers further advanced nano-DESI with a technique they called pneumatically assisted nano-DESI [64], which has a similar relationship to nano-DESI as AFADESI has to DESI; pneumatically assisted nano-DESI allows for remote sampling of analytes at greater sensitivity. In pneumatically assisted nano-DESI, a nebuliser gas inlet is introduced to the secondary (nanospray) capillary. By directly introducing a gas flow through the nanospray capillary the probe can be placed further from the inlet and there is an enhanced sensitivity for metabolite species detection and a lower effect of probe-to-sample surface distance on the analyte signal intensity [64]. Hybridisation of DESI to other sources and analysers has also introduced new modalities for analysis. Wood and coworkers utilised an FT-ICR mass spectrometer equipped with both a DESI and MALDI source, generating increased lipid coverage of rat brain than either source individually, without the need to switch ion sources [54]. Pan and coworkers combined a standard DESI source with a photoionisation source to analyse lipids in mouse brain, revealing an increased number of identified lipids, in particular non-polar lipids, in both positive and negative mode compared to DESI alone [67]. Laskin and coworkers incorporated shear force microscopy into a nano-DESI probe, allowing for the probe to be kept a constant height from the substrate surface even when complex topography is present [68][69][70].

Laserspray ionisation is a technique introduced by Trimpin et al. in
2010 with the aim of providing ESI-like spectra using standard MALDI sample preparation conditions [93]. In summary, a matrix/analyte mixture is co-crystallised onto a sample target that is placed in front of a heated ion transfer tube, which transfers the laser-desorbed sample material to the mass spectrometer inlet. Both AP and vacuum sources have been used for laserspray ionisation. In contrast to standard (AP-)MALDI and LAP-MALDI (see below), laserspray ionisation employs much higher laser fluences (≥10 kJ/m 2 ). Initially used to investigate the formation of gas-phase ions with MALDI-like techniques, the technique was also used with ETD [93], for in situ protein analysis [94] and MSI [95]. However, not much research was recently published with this technique, potentially due to current advances in DESI and other AP-MALDI techniques, being less destructive in their approach.

AP-MALDI
MALDI MS has long been one of the techniques of choice for automated high-throughput proteomics, due to its excellent sensitivity and analysis speed compared to other proteomic MS techniques [96]. It has been recognised as a complementary tool to ESI for proteomics analyses, particularly when coupled to LC [97].  [101]. Further optimisation of the heated ion transfer tube and gas counterflow during ion transfer led to a 14-fold increase in the ion yield of multiply protonated peptide analytes [102,103]. Collisioninduced dissociation (CID), a molecular fragmentation technique that improves in efficacy and sensitivity with an increase in charge state, was utilised with the LAP-MALDI setup to great effect, in structural lipidomics [104], protein/peptide identification [105] and disease diagnostics in combination with simultaneous multi-omic profiling [106].
Multi-omic profiling of a variety of sample types has been further developed and applied to microbial biotyping [107], speciation [108] and the early and cost-effective detection of mastitis from crude milk samples [109]. Owing to the high-throughput capabilities [106,109,110] of the technique combined with its extremely stable ion signal as well as new data acquisition software, record analysis speeds of up to 60 samples per second have been achieved [110,111].

Rapid evaporative ionisation MS (REIMS)
REIMS enables direct sampling of difficult to handle samples and substrates such as biological tissues and fluids. First described in 2009 by Schäfer et al. [112], the technique originally employed an electro-surgical electrode that makes contact with the sample, rapidly evaporating biological material to generate gaseous molecular ions such as phospholipids [112,113]. As part of the 'iKnife' technology, REIMS has demonstrated to be a hybrid surgical/analytical tool that can profile biological tissue during surgery [113][114][115], as well as rapidly profile the metabolome and lipidome of various crude biological fluids, like faeces, bile and urine [116]. where the metabolomic profiles of normal weight and obese individuals were successfully differentiated [119]. However, these applications have all the same potential limitations of poor quantitation and ion suppression due to the lack of prior sample preparation and therefore matrix effects [120], which on the other hand provides simplicity, universality and speed [121].

Acoustic ionisation techniques
One class of (AP) ionisation techniques that has seen more popularity in recent years is acoustic ionisation. The energy of acoustic waves typically produced by a transducer leads to the ejection of small amounts of liquid sample material from a well, allowing for rapid sample analyses from microtiter plates with little sample consumption making these techniques ideal for screening and profiling studies [122] (1) to initiate microdroplet formation from the sample liquid in the microtiter plate well (2). Droplets are formed at the sample's surface (3); their formation and analyte ionisation is supported by a high voltage applied to a cone in front of the mass spectrometer inlet (4). The microdroplets pass through a heated ion transfer tube (5) to promote desolvation, and finally towards the mass spectrometer inlet (6). Adapted with permission from ref. [124]. Copyright 2019 American Chemical Society rate of >10,000 data points per hour, making it an excellent technique for high-throughput analysis. Later studies referred to this type of technique as acoustic mist ionisation (AMI) and applied it to enzymatic assay development for high-throughput biochemical screening, demonstrating more than 100,000 samples per day [124]. to ADE, efficiently collecting and diluting the acoustically ejected (nL-)droplets in a solvent flow, which is then delivered to an ESI source [20,127]. Various applications, from drug-drug interaction to pharmacokinetic and biomarker analysis, have been demonstrated [127].
Although this setup is also applicable to high-throughput analysis, its ultimate sampling rate is limited by sample diffusion within the transfer capillary to the ESI source.

Matrix-assisted ionisation vacuum (MAIV)
Another relatively new technique is MAIV, first described by Trimpin and Inutan in 2013 [130]. MAIV is an ionisation method that applies no laser irradiation or high voltages, and simply requires an analyte/matrix (typically 3-nitrobenzonitrile) mixture to be placed against the inlet cone of the mass spectrometer held at a vacuum. In one of the original publications, different methods of introducing the sample to the inlet cone were investigated, with equivalent ion counts between a KimWipe, the pointed tip of a strip of filter paper (similar to paper spray ionisation but without the high voltage), and a pipette tip [130]. +18 charge state. The in situ analysis of peptides and proteins from rat brain tissue extracts were also performed, with a variety of singly charged and multiply charged peptides and proteins detected [132].
Most recently, Harding et al. were able to perform lipidomic MSI experiments of rat brain tissue using MAIV, optimising sample extraction into the 3-nitrobenzonitrile matrix by the addition of 5% chloroform [133].

CONCLUDING REMARKS
In this review, the landscape of MS ionisation techniques newly introduced for omics analyses over recent years has been examined.  MALDI matrices for low molecular weight compounds: An endless