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Updated: May 18, 2026
Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows
Published on: January 16, 2018
An optimized matrix-assisted laser desorption/ionization sample preparation using a liquid matrix,
Yuko Fukuyama1, Kohei Takeyama, Shin-ichirou Kawabata
1Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto, 604-8511, Japan. yukof@shimadzu.co.jp
Rationale:
A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides.
Methods:
We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic acid (2,5-DHB).
Results:
The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels.
Conclusions:
Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics.
