A Response Surface Methodology Framework for Targeted MALDI‑TIMS Optimization and in‑Tissue Metabolite Resolution
- 1Integrated Discovery Sciences Directorate
- 2Pacific Northwest National Laboratory
- 3Indiana University School of Medicine
Abstract
Differentiating hexose‑phosphate isomers directly within tissue is essential for understanding glycolytic and pentose‑phosphate pathway activity, particularly in the kidney, where these metabolites play central roles in energy balance, redox regulation, and stress response. However, their high structural similarity and low endogenous abundance make on‑tissue discrimination analytically challenging. To address this, we systematically optimized MALDI‑TIMS‑TOF parameters using response surface methodology (RSM), developing a predictive model that revealed positive linear and negative quadratic effects of ramp time, a positive linear dependence on TIMS pressure, no significant contribution from the mobility window, and no factor interactions. Balancing analytical sensitivity and mobility resolution identified 720 ms ramp time and 2.5 mbar TIMS pressure as optimal conditions for targeted hexose‑phosphate separations. Under these optimized conditions, MALDI‑TIMS MSI of mouse kidney with lipopolysaccharide (LPS)-induced injury uncovered an endogenous feature with the same nominal mass as hexose phosphates but with mobility distinct from all tested standards, including glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate and mannose-6-phosphate. This molecule showed differential localization between cortex and medulla and responded to LPS‑induced metabolic stress, revealing unanticipated biochemical heterogeneity within the hexose‑phosphate mass range. Together, these findings demonstrate that RSM‑guided TIMS optimization enhances the resolving power of mobility‑based separations and expands the discovery potential of MALDI‑TIMS MS for uncovering molecular complexity directly within biological tissues. INTRODUCTION Sugar phosphates are central intermediates that connect glycolysis, gluconeogenesis, the pentose phosphate pathway, and glycogen metabolism, thereby sustaining energy production and redox balance in mammalian tissues1. In the kidney—one of the body’s most metabolically demanding organs—hexose phosphates play essential roles in proximal tubular glucose handling and phosphate‑dependent metabolic regulation2. Among these metabolites, glucose‑6‑phosphate (Glc6P) links glycolysis to NADPH‑producing pathways important for maintaining tubular antioxidant defense3, fructose‑6‑phosphate (Fru6P) modulates carbohydrate flux in renal epithelial cells2, and glucose‑1‑phosphate (Glc1P) contributes to glycogen turnover and metabolic buffering during renal stress4, 5. Perturbations in these tightly regulated metabolites accompany inflammation and acute kidney injury1, 6, 7, underscoring the need for analytical strategies that can distinguish sugar‑phosphate isomers directly in tissue. Differentiating sugar‑phosphate isomers remains analytically challenging because their high polarity and minimal structural differences limit the resolving power of conventional approaches. Chromatographic strategies—including hydrophilic interaction liquid chromatography (HILIC),
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BibTeX
@misc{karkamkar2026response,
title = {{A Response Surface Methodology Framework for Targeted MALDI‑TIMS Optimization and in‑Tissue Metabolite Resolution}},
author = {Keshav Karkamkar and Heidi Vandyk and Azuma Nanamatsu and Tarek M El-Achkar and Dušan Veličković},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010128/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010128/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A Response Surface Methodology Framework for Targeted MALDI‑TIMS Optimization and in‑Tissue Metabolite Resolution
AU - Keshav Karkamkar
AU - Heidi Vandyk
AU - Azuma Nanamatsu
AU - Tarek M El-Achkar
AU - Dušan Veličković
PY - 2026
DA - 2026/10/08
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010128/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010128/v1
AB - Differentiating hexose‑phosphate isomers directly within tissue is essential for understanding glycolytic and pentose‑phosphate pathway activity, particularly in the kidney, where these metabolites play central roles in energy balance, redox regulation, and stress response. However, their high structural similarity and low endogenous abundance make on‑tissue discrimination analytically challenging. To address this, we systematically optimized MALDI‑TIMS‑TOF parameters using response surface methodology (RSM), developing a predictive model that revealed positive linear and negative quadratic effects of ramp time, a positive linear dependence on TIMS pressure, no significant contribution from the mobility window, and no factor interactions. Balancing analytical sensitivity and mobility resolution identified 720 ms ramp time and 2.5 mbar TIMS pressure as optimal conditions for targeted hexose‑phosphate separations. Under these optimized conditions, MALDI‑TIMS MSI of mouse kidney with lipopolysaccharide (LPS)-induced injury uncovered an endogenous feature with the same nominal mass as hexose phosphates but with mobility distinct from all tested standards, including glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate and mannose-6-phosphate. This molecule showed differential localization between cortex and medulla and responded to LPS‑induced metabolic stress, revealing unanticipated biochemical heterogeneity within the hexose‑phosphate mass range. Together, these findings demonstrate that RSM‑guided TIMS optimization enhances the resolving power of mobility‑based separations and expands the discovery potential of MALDI‑TIMS MS for uncovering molecular complexity directly within biological tissues. INTRODUCTION Sugar phosphates are central intermediates that connect glycolysis, gluconeogenesis, the pentose phosphate pathway, and glycogen metabolism, thereby sustaining energy production and redox balance in mammalian tissues1. In the kidney—one of the body’s most metabolically demanding organs—hexose phosphates play essential roles in proximal tubular glucose handling and phosphate‑dependent metabolic regulation2. Among these metabolites, glucose‑6‑phosphate (Glc6P) links glycolysis to NADPH‑producing pathways important for maintaining tubular antioxidant defense3, fructose‑6‑phosphate (Fru6P) modulates carbohydrate flux in renal epithelial cells2, and glucose‑1‑phosphate (Glc1P) contributes to glycogen turnover and metabolic buffering during renal stress4, 5. Perturbations in these tightly regulated metabolites accompany inflammation and acute kidney injury1, 6, 7, underscoring the need for analytical strategies that can distinguish sugar‑phosphate isomers directly in tissue. Differentiating sugar‑phosphate isomers remains analytically challenging because their high polarity and minimal structural differences limit the resolving power of conventional approaches. Chromatographic strategies—including hydrophilic interaction liquid chromatography (HILIC),
ER -