FlexiFocus ™ Slides: Chemically Modified Sample-Focussing MALDI Targets to Enhance Productivity and Throughput

Importance of the Topic

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a cornerstone technique in proteomics, lipidomics, oligonucleotide analysis and quality control applications.

Precise sample spot alignment and consistent crystallization are essential for reproducible automated analysis and optimal sensitivity in high-throughput workflows.

Chemically modified targets that guide sample droplets to a defined hydrophilic region can minimize signal variability and support robust data acquisition.

Aims and Overview of the Study

This work presents FlexiFocus™ slides, a novel MALDI target with a hydrophobic coating surrounding micrometer-scale hydrophilic wells designed to self‐centre deposited samples.

The study evaluates sample containment, spot focusing behavior, sensitivity enhancement and compatibility with common matrices, solvents and automated platforms.

Comparisons are made to standard stainless steel FlexiMass-SR48 targets to demonstrate improvements in throughput and detection limits.

Used Instrumentation

• Shimadzu MALDI-8020 benchtop MALDI-TOF mass spectrometer
• Shimadzu FlexiFocus™ 48- and 96-well slide inserts with magnetic adaptors
• Standard FlexiMass-SR48 stainless steel targets for benchmarking
• Shimadzu QC Reporter software for automated quality control assays

Methodology

FlexiFocus slides feature a metal substrate coated with a highly hydrophobic layer interrupted by exposed 900 µm hydrophilic spots in each well.

Sample deposition experiments tracked droplet migration and drying kinetics at defined time points to compare focusing efficiency.

Representative analytes included immunoglobulin A (IgA), triacylglycerols (TAGs) from extra‐virgin olive oil, Glu1-fibrinopeptide B dilutions, a 20-mer oligonucleotide and a bovine serum albumin (BSA) tryptic digest.

Procedures varied matrix type and concentration (CHCA, sinapinic acid, DHB, 3-HPA), solvent composition and analyte amounts down to tens of attomoles on target.

Protein identification was confirmed by peptide mass fingerprinting (Mascot PMF), oligonucleotide sequence coverage assessed by in-source decay, and QC outcomes recorded in QC Reporter.

Main Results and Discussion

Spot focusing studies showed off-center droplets migrate to the central hydrophilic zone on FlexiFocus slides, whereas FlexiMass-SR48 spots dried in their original position, leading to misalignment.

Analysis of IgA (5 pmol) yielded clear monomer (~160 kDa), doubly charged (~80 kDa) and heavy chain (~55 kDa) peaks with standard crystal morphology.

TAG profiling of olive oil at 5 mg/mL identified major species (OOO, OOP, OOL, POL) with full isotopic resolution.

Peptide sensitivity tests detected GluFib down to 25 amol on target with signal‐to‐noise ratios >3 and resolved isotopic envelopes.

A 20-mer oligonucleotide prepared at 100 µM produced intact [M+H]+ at m/z 6118 and achieved 75 % sequence coverage by MALDI-ISD.

BSA digest detection limits improved such that high‐confidence Mascot IDs were obtained at 100 amol on FlexiFocus compared to 300 amol on FlexiMass-SR48; further enhancement was seen at 30 amol with increased laser shots and matrix additives.

QC workflows on a FlexiFocus-96 slide showed reliable PASS/QUERY/FAIL classification of synthetic peptide controls in QC Reporter.

Benefits and Practical Applications

• Enhanced spot centering ensures consistent laser sampling and reduces signal dropouts in automated runs.
• Improved sensitivity lowers sample consumption for low-abundance analytes in proteomics and lipidomics.
• Broad compatibility with standard MALDI matrices and solvents simplifies integration into existing protocols.
• Adaptors and slide formats support both 48- and 96-well high-throughput analyses on benchtop MALDI-TOF platforms.
• Seamless operation with QC Reporter streamlines routine quality control testing in regulated environments.

Future Trends and Potential Applications

Integration of microfabricated hydrophilic patterns with multiplexed MALDI imaging or ion mobility separations may further boost spatial resolution and analytical depth.

Development of new surface chemistries could extend sample focussing to aqueous biofluids, nanoparticles or complex matrices.

Coupling FlexiFocus technology with emerging ultra-high-throughput MALDI-TOF/TOF and AI-driven data analysis promises accelerated biomarker discovery and translational research.

Conclusion

FlexiFocus slides deliver robust sample containment and automatic spot centering that enhance sensitivity, reproducibility and throughput in MALDI-TOF workflows.

Their compatibility with diverse matrices, solvents and automated QC software makes them a versatile tool for proteomics, lipidomics, oligonucleotide analysis and routine quality control.

Reference

• Salivo S. FlexiFocus™ Slides: Chemically Modified Sample-Focussing MALDI Targets to Enhance Productivity and Throughput. Shimadzu Application Note C146-E479, First Edition October 2023.