High‐Throughput N‐Glycan Analysis by IR‐MALDESI With a 1.5‐kHz Pulsed Mid‐IR Laser

Seth M Eisenberg; Sarah M Ashbacher; Alexander A C Wainwright; Aosheng Gu; R J Dwayne Miller; David C Muddiman

doi:10.1002/jms.70056

. 2026 Apr 7;61(5):e70056. doi: 10.1002/jms.70056

High‐Throughput N‐Glycan Analysis by IR‐MALDESI With a 1.5‐kHz Pulsed Mid‐IR Laser

Seth M Eisenberg ¹, Sarah M Ashbacher ¹, Alexander A C Wainwright ², Aosheng Gu ², R J Dwayne Miller ^2,^3,^✉, David C Muddiman ^1,^✉

PMCID: PMC13055878 PMID: 41945006

ABSTRACT

Infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) is a laser‐based, hybrid ionization source for mass spectrometry, enabling both mass spectrometry imaging (MSI) and high‐throughput screening (HTS). In this work, a 2.2‐ns, 2.72‐μm laser, operating at 1.5 kHz, was integrated into the IR‐MALDESI platform to evaluate its performance for robust HTS and glycan profiling. The system produced robust HTS performance, achieving a relative standard deviation (RSD) of 9.1% with no detectable carryover between wells and consistent ion abundances in multiwell screening experiments. Using this configuration, 29 glycans were detected from a solution of cleaved N‐glycans derived from bovine fetuin, with multiple charge states observed for several species. These results demonstrate rapid, high‐throughput analysis and expanded glycan detection enabled by desorption using a low‐power mid‐IR laser operating at kHz repetition rates, highlighting its potential for advanced biochemical screening and characterization.

Keywords: glycans, high‐throughput screening, IR‐MALDESI, mid‐IR laser, nanosecond laser

1. Introduction

High‐throughput screening (HTS) enables rapid evaluation of large numbers of samples and has become a cornerstone technique in modern analytical and pharmaceutical research [1, 2, 3]. Traditionally, HTS has been employed by pharmaceutical companies to identify potential drug candidates, where each sample is assessed for interaction with a target of interest. Among the many detection methods available, mass spectrometry (MS) is increasingly favored because of its high sensitivity, specificity, and ability to detect a broad range of analytes [4]. Ambient MS techniques have expanded the capabilities of HTS by eliminating the need for extensive sample preparation and have demonstrated low sensitivity to salts and complex matrices [5, 6, 7]. These features allow for faster and more flexible screening compared to traditional LC–MS workflows. Several types of ionization sources are commonly used for HTS applications, including spray‐based methods (e.g., desorption electrospray ionization [DESI]) [8, 9], electric discharge‐based techniques (e.g., direct analysis in real time [DART]) [10, 11], and laser‐assisted desorption/ionization methods (e.g., infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI)) [12, 13].

While HTS has been widely applied to drug discovery and metabolite screening, the same high‐throughput capabilities can also accelerate the analysis of complex biomolecular modifications such as glycosylation, where sample diversity and structural complexity often limit traditional workflows [14]. Glycosylation is one of the most common posttranslational modifications (PTMs) observed in nature, wherein proteins undergo the addition of various sugars through the endoplasmic reticulum and Golgi apparatus [15]. Among these, N‐linked glycans share a conserved pentasaccharide core and are formed at specific amino acid motifs [16]. These structures are involved in a range of biological processes including protein folding, cell signaling, proliferation, and adhesion [17, 18]. Aberrant glycosylation patterns have been associated with a wide range of diseases and serve as potential biomarkers for cancer [18, 19, 20, 21], as well as Alzheimer's disease [22], diabetes, and various autoimmune disorders [23, 24]. Glycosylation profiles are also routinely monitored as critical quality attributes in the drug development and manufacturing process, as they significantly impact immunogenicity, pharmacokinetics and dynamics, and overall drug efficacy [25, 26, 27, 28]. These factors highlight the growing need for faster, high‐throughput glycan profiling techniques.

Several ambient MS platforms have demonstrated potential for rapid glycan and biomolecule screening. DESI enables analysis of reaction droplets at rates approaching one sample per second [8] and has recently been utilized for ligand screening to identify high‐affinity protein binders [29, 30]. DART allows rapid screening of over 50 drugs in 1 min [10] and has been applied to steroid identification and quantification [11]. MALDI‐MSI has been used for serum glycoprotein analysis, detecting over 75 N‐glycans within approximately 7 h [31], and over 100 distinct N‐glycans in other studies [32]. However, MALDI‐based approaches often require chemical derivatization, such as permethylation or esterification, to prevent sialic acid loss from labile glycan residues. In contrast, IR‐MALDESI combines laser ablation with electrospray ionization to achieve high‐throughput screening speeds, reaching up to 22.7 samples per second, enabling a 384 well plate to be analyzed in just 17 s [7]. Notably, IR‐MALDESI enables the direct detection of intact, sialylated N‐glycans without derivatization [33].

Recently, low‐power, kilohertz (kHz) repetition rate, single pulse mid‐IR lasers with short pulse durations have become commercially available and been applied to IR‐MALDESI MSI and HTS applications [34]. While initial demonstrations have shown promise [35], further optimization and evaluation are required. In this work, a commercial 2.2‐ns, 2.72‐μm laser with a pulse energy of 25 μJ operating at 1.5 kHz was incorporated into the IR‐MALDESI system with a remote‐controlled shutter to regulate the number of laser pulses per mass spectrum. This configuration was used to assess reproducibility between scans and carryover between wells, followed by application to N‐linked glycan analysis, demonstrating the detection of N‐glycans from bovine fetuin. Together, these developments establish a foundation for highly reproducible, fast glycan analysis by IR‐MALDESI, highlighting its potential as a next‐generation platform for high‐throughput biochemical and biopharmaceutical screening.

2. Experimental

2.1. Materials

LC–MS grade water, acetonitrile (ACN), formic acid, and acetic acid were purchased from Thermo Fisher Scientific (Nazareth, PA, USA). Nitrogen gas was purchased from Arc3 gases (Raleigh, NC, USA).

2.2. Sample Information

For high‐throughput analysis, a 384 well plate was prepared in a checkerboard pattern by alternating wells containing analyte solution and water, respectively. Wells contained 30 μL of solutions of 5 μM caffeine, 3 μM glutathione, 0.3 mM fluconazole, and a peptide mixture containing 18 mM leu‐enkephaline, 8.46 mM gonadoliberin, 7.71 mM angiotensin I, and 5.98 mM neurotensin that were dissolved in LC–MS grade water. These solutions were subsequently analyzed by IR‐MALDESI to evaluate sampling speed, %RSD, and cross‐contamination between adjacent wells.

Bovine fetuin glycans were prepared as previously described [36]. In brief, 250 μg of bovine fetuin, dithiothreitol, and 100 mM ammonium bicarbonate were combined in a 10 kDa molecular weight cutoff filter. Following denaturation, the proteins were alkylated with iodoacetamide and glycans were cleaved by adding 1000 units of PNGase F PRIME‐LY (Bulldog Bio, NJ, USA) directly into the filter and incubating overnight at 37°C. The released glycans were eluted, dried in a vacuum desiccator, and resuspended in LC–MS grade water.

2.3. Pulse Mode 1.5‐kHz Laser and Setup

The pulse mode laser (IVY, Glucoloop AG, Zurich, Switzerland) operates at a wavelength of 2.72 μm, enabling selective excitation of O‐H stretching bands of water. It emits 2.2‐ns pulses at a repetition rate of 1.5 kHz. The laser is diffraction limited, allowing precise focusing, with each pulse delivering 25 μJ of energy and a peak power of 11 kW.

The pulsed 1.5‐kHz laser operating at 25 μJ per pulse was integrated into the IR‐MALDESI apparatus. The laser beam was routed through a remote‐controlled shutter (Thorlabs, Cat #SH1) connected to a programmable controller (Thorlabs, Cat #SC30), which allowed for both manual and automated triggering. Beam steering was accomplished using adjustable mirrors, followed by elevation via a periscope to a 90° mirror, directing the laser downward into the IR‐MALDESI enclosure. The downtube incorporated a 2.5‐mm iris followed by a reflective objective [37] (Thorlabs, Cat #LMM15X‐PO1) to focus the beam onto the sample surface. Using this optical configuration, portrayed in Figure 1, the measured pulse energy at the sample surface was 13 μJ/pulse.

Diagram detailing the optical configuration from the laser to the IR‐MALDESI enclosure. The laser beam was fired at 1.5 kHz and controlled by an automatic shutter before entering steering mirrors and eventually being focused using a reflective objective.

2.4. IR‐MALDESI‐MSI Analysis

The next‐generation IR‐MALDESI source was employed for droplet and well plate experiments as previously described [38]. After ionization, analytes were measured using an interfaced Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [39]. The laser was operated at 2.72 μm to resonantly excite the O‐H stretching vibrational modes of the solution, similar to previous works [38, 40, 41, 42, 43, 44]. A remote‐controlled shutter was used to precisely regulate the number of laser pulses delivered to the sample during each mass spectrometer scan. The molecules generated from laser ablation are partitioned into the orthogonal electrospray plume and subsequently ionized via an electrospray‐like mechanism. In positive mode, the electrospray solvent consisted of 60% ACN with 0.2% formic acid in water, delivered at a flow rate of 1.2 μL/min with an applied voltage of 3.6 kV [45]. In negative mode, the electrospray solvent was made of 50% ACN with 1 mM acetic acid in water at 1.2 μL/min and −3.6 kV of applied voltage.

Mass spectrometric data were acquired in positive ion mode for well‐plate analysis and negative ion mode for glycan analysis. All experiments were performed at a resolving power of 240 000 (FWHM at m/z 200) with the EASY‐IC internal calibrant fluoranthene ([M^+●] m/z 202.0777) enabled. Automatic gain control was disabled, and the injection time was held constant at 15 ms for well‐plate analysis and 90 ms for glycans, respectively. Mass spectra were recorded over variable m/z ranges depending on the application, with the S‐lens RF value set to 70% consistently.

2.5. Data Analysis

The raw mass spectra were analyzed using XCalibur (Thermo Fisher Scientific). All optical images were taken using a Leica LMD 7000 microscope (Leica Microsystems, Wetzlar, Germany). Glycans were identified by uploading to GlycoMod, a theoretical glycan database that can generate N‐linked glycan annotations. GlycoMod also provides a list of previously reported glycans through GlyConnect. Additionally, glycan annotations were directly compared against lists of previously detected glycans in bovine fetuin by IR‐MALDESI [36].

3. Results and Discussion

The performance of the 1.5‐kHz laser was evaluated using a well plate experiment (Figure 2). In this setup, a dilute standard solution was analyzed, followed by acquisition of a background ESI spectrum, and finally a blank measurement from a well containing pure HPLC‐grade water. This experimental design allowed for assessment of the laser's reproducibility and consistency by examining signal variability over approximately 200 scans of a homogenous solution. Additionally, the potential for carryover between wells was investigated, as unintended splashing or mixing caused by laser firing could confound analytical results. Carryover was evaluated by comparing analyte signals in the blank water wells to the background abundance measured without laser irradiance.

(A) Experimental setup for well‐plate analysis by IR‐MALDESI. (B) Well contains (9A) 5.15 μM caffeine, (11A) 3.25 μM glutathione, (13A) 0.33 mM fluconazole, (15A) peptide mix containing 18.0 mM leu‐enkephalin, 8.46 mM gonadoliberin, 7.71 mM angiotensin I, and 5.98 mM neurotensin. Wells 10A, 12A, 14A, and 16A contain LC–MS grade water. Analytes showed no carryover between wells.

The results demonstrated that no carryover occurred between wells, indicating that the low pulse energy of the laser did not induce inter‐well contamination. This feature is particularly advantageous for high‐throughput screening, as it ensures rapid, sequential analysis of wells without introducing cross‐contamination or false‐positive results.

It should be noted that in this initial experiment, percent relative standard deviation (%RSD) and signal variability were not quantified because the shutter and ion injection time were not synchronized, resulting in differing number of laser shots per scan. In subsequent experiments, synchronization of the shutter and MS injection time allowed for a consistent number of laser pulses per scan, yielding a %RSD of 9.1%, consistent with previously reported results [35]. These findings confirm that using single pulse lasers with repetition rates below 10 kHz and slightly detuned from the O‐H stretching mode of water (2.94 μm) delivers highly repeatable ion abundance, underscoring its suitability for robust, high‐throughput analyses. This is particularly significant because developing fiber‐based mid‐IR lasers from 2.7 to 2.85 μm is considerably easier than at 2.94 μm, as materials that lase at these wavelengths are readily available [46, 47]. These lasers are optimized for systems requiring kHz repetition rates, which we have shown are advantageous for HTS applications. These advantages lead to lower production costs and broader adoption of platform technology.

Following the successful demonstration of the 1.5‐kHz laser and well‐plate setup for HTS using standard solutions, cleaved N‐linked glycans from bovine fetuin were analyzed under the same experimental conditions. A total of 29 distinct glycans were confidently identified from this single glycoprotein, listed in Supplemental Information (Table S1), reflecting broad glycan coverage and robust spectral quality. Notably, 12 glycans detected were not previously observed by previous iterations of the IR‐MALDESI system (Table 1). A representative mass spectrum obtained with the 1.5‐kHz laser is shown in Figure 3, illustrating strong ion abundances routinely achievable across diverse glycan compositions. The high signal levels provide improved isotopic clarity, enable confident assignment of low abundance glycans, and support observation of multiply charged species.

TABLE 1.

List of N‐linked glycans detected using the 1.5‐kHz laser that have not been previously observed by IR‐MALDESI. Glycans marked as potential have been observed and characterized but not detected in bovine fetuin glycoprotein in literature.

Glycoform mass	Structure	[M‐2H]²⁻	[M‐3H]³⁻
1233.4494	Hex₅HexNAc₂	615.7174	Not detected
1395.5022	Hex₆HexNAc₂	696.7438	Not detected
1598.5816	Hex₆HexNAc₃	798.2835	Not detected
1727.6242	Hex₅HexNAc₃NeuAc₁	862.8048	Not detected
1889.677	Hex₆HexNAc₃NeuAc₁	943.8312	Not detected
1930.7036	Hex₅HexNAc₄NeuAc₁	964.3445	Not detected
2076.7615	Hex₅HexNAc₄Fuc₁NeuAc₁	1037.3735	Not detected
2239.8216	Hex₆HexNAc₄Fuc₁NeuAc₁	1118.3999	Not detected
2311.6987	Hex₃HexNAc₃dHex₁Sulph₁ (potential)	1154.8415	Not detected
2383.8518	Hex₆HexNAc₄NeuAc₂	1190.9186	Not detected
2894.0467	Hex₅HexNAc₃Fuc₄NeuAc₃	1446.0161	963.6749
3259.0243	Hex₄HexNAc₄dHex₁NeuAc₂Sulph₂ (potential)	Not detected	1085.3336

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Mass spectrum showing the abundances of representative glycans with their respective *m/z* value, charge state, and glycan structure and identification.

Figure 4 highlights representative glycans demonstrating high spectral accuracy (χ² < 5.991 at n = 3), mass measurement accuracy (< 2.5 ppm), and well‐resolved isotopic distributions, confirming putative identifications and the preservation of labile functional groups, such as sialic acids and sulfates.

Spectral accuracy plots of four representative glycans. The red dots indicate the theoretical abundance values and the spectra indicates the measured abundances. Chi‐squared values are shown for each glycan. The values were compared to the critical value of 5.991 for 3° of freedom. For consistency, 3° of freedom was utilized for the four comparisons. These results indicate nonsignificant differences between theoretical and measured spectral accuracy.

The 2.2‐ns pulses from the 1.5‐kHz laser fall within the desorption by impulsive vibrational excitation (DIVE) regime, in which rapid vibrational energy deposition drives molecules to exceed their binding energies before heat or pressure can dissipate (Figure 5) [48, 49]. This ultrafast vibrational loading produces efficient release of material into the gas phase, thereby supporting efficient ionization by the intersecting electrospray and contributing to the strong mass spectral responses obtained under these conditions. In addition to the short pulse duration, the high number of pulses per acquisition (135 pulses during the 90‐ms acquisition period) likely promotes cumulative desorption of material into the ionization region. Successive nanosecond pulses can continue to interact with the sample within the irradiated area, maintaining consistent desorbed species throughout the acquisition period. This sustained production of gas phase analytes aligns with the stable ion populations observed throughout the experiment.

Proposed mechanism of desorption by impulsive excitation (DIVE). A short IR laser pulse delivers energy to the sample, exciting the O‐H stretching vibrational mode of water. The pulse is shorter than the acoustic relaxation time, resulting in the thermal energy being confined to the irradiated area, resulting in precise ablation. This results in a rapid vaporization occurring within a few nanoseconds, creating a plume of molecules that intersect with the electrospray.

Plume‐dynamics studies using similar ns mid‐IR irradiation provide further mechanistic context. Shadowgraph and dark‐field imaging of nanosecond and picosecond mid‐IR laser desorption of water show that large droplets appear later in the process [50], whereas vapor is produced at the start of desorption by shorter pulses within the DIVE regime. These established behaviors of ns‐pulse mid‐IR ablation support the interpretation that short‐pulse excitation within the DIVE regime favors efficient production of analytes, while plume dynamics can influence the degree of interaction between subsequent pulses and the ejected material.

The performance of the 1.5‐kHz laser significantly expands the analytical capabilities of IR‐MALDESI for glycan analysis. These advances not only facilitate the detection of glycoforms in multiple charge states but also show the potential for increased throughput, making this approach well‐suited for a wide range of applications, including glycan profiling, protein‐ligand interaction studies, and broader high‐throughput drug screening workflows.

4. Conclusions

The short pulsed, 1.5 kHz mid‐IR laser at 2.72 μm was successfully integrated into the IR‐MALDESI platform and demonstrated HTS of standard solutions and N‐linked glycans. When combined with a remote‐controlled shutter to precisely regulate the number of pulses per scan, the system exhibited high reproducibility, achieving a %RSD of ~9% across multiple scans. Application to cleaved N‐linked glycans from bovine fetuin resulted in the detection of a total of 29 distinct glycoform masses that are doubly and triply charged. Nanosecond lasers operating at kHz repetition rates and tuned to the O‐H absorption band of water significantly improve high‐throughput glycan analysis and other HTS applications. In the future, the glycan profiles generated may be supplemented by glycopeptide detection following tryptic digestion.

Funding

This work was supported by the National Institute of General Medical Sciences (R01GM087964) and the Natural Sciences and Engineering Research Council of Canada.

Conflicts of Interest

David C. Muddiman is a partial‐owner of MSI Software Solutions LLC. The authors declare no conflicts of interest.

Supporting information

Table S1: List of N‐linked glycan mass, structures, and detected adducts. Glycan structures marked as potential have been identified in databases but not observed in the bovine fetuin glycoprotein. Glycan structures marked as unknown were glycans not given a theoretical structure by GlycoMod.

JMS-61-e70056-s001.docx^{(37.9KB, docx)}

Acknowledgements

The authors gratefully acknowledge financial support from the National Institutes of Health (R01GM087964) and the Natural Sciences and Engineering Research Council of Canada (R.J.D.M.). All mass spectrometry measurements were conducted at the Molecular Education, Technology, and Research Innovation Center (METRIC) at the NC State University.

Contributor Information

R. J. Dwayne Miller, Email: dwayne.miller@utoronto.ca.

David C. Muddiman, Email: dcmuddim@ncsu.edu.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

JMS-61-e70056-s001.docx^{(37.9KB, docx)}

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

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PERMALINK

High‐Throughput N‐Glycan Analysis by IR‐MALDESI With a 1.5‐kHz Pulsed Mid‐IR Laser

Seth M Eisenberg

Sarah M Ashbacher

Alexander A C Wainwright

Aosheng Gu

R J Dwayne Miller

David C Muddiman

ABSTRACT

1. Introduction