Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging

Abstract

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25-30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60-75% ACN/40-25% H₂O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45-55% ACN/55-45% H₂O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

Keywords: IR-MALDESI; lipids; mass spectrometry imaging: electrospray ionization; metabolites.

Figure 1

ACN, MeOH gradients were run from 5—95% with voltages from 2.25 to 4.5 kV in both positive and negative polarities

Figure 2

Onset voltages predictions for IR-MALDESI (black dots) change with solvent composition (red dots). Measured onset voltages in IR-MALDESI in positive and negative polarities at both 5:95 and 95:5 water:solvent endpoints are shown as stars in red (positive) and blue (negative)

Figure 3

(a) Total ion current (TIC) of 85 tissue specific lipids plotted across 5–95% ACN gradients for voltages from 2.25 kV to 4.5 kV (blue) and percent coverage of those lipids (green). Percent coverage is defined as number of lipids detected/85 total assignable lipids. (b) TIC of tissue specific lipids plotted as a ratio of the Total TIC across voltages to determine which resulted in greater relative abundance of the lipids. (c,d) Panels are for MeOH

Figure 4

Relative abundances of 85 tissue specific lipids across the gradients of (a) ACN and (b) MeOH. They are shown from low to high m/z from left to right across each image. Each column contains abundance data that is relatively scaled per lipid and plotted linearly from black (low) to red (middle) to yellow (high) to determine which gradient range was best for each lipid.

Figure 5

(a, d) The TIC of 85 tissue specific lipids is plotted in blue and percent coverage of those lipids is in green. (b, e) The TIC of tissue specific lipids is plotted as a ratio of the whole TIC. (c, f) The lipid abundances are plotted as mentioned in Figure 4, except they are shown from low to high m/z from top to bottom of each image. a, b, c represent the ACN gradients, and d, e, f represent MeOH gradients

Figure 6

Relative abundances of 133 tissue specific lipids found in both gradients of ACN (a) and MeOH (b). They are shown from low to high m/z from left to right across each image. Each column contains abundance data. Abundance per lipid is plotted linearly from black (low) to blue (middle) to yellow (high) to determine which gradient range was best per lipid and for the combined group

Figure 7

(a) Total ion current (TIC) of 133 assignable tissue specific lipids plotted across ACN gradients for voltages from 2.25 kV to 4.5 kV (blue) and percent coverage of those lipids (green). Percent coverage is defined as number of lipids measured divided by 133 assignable lipids. (b) TIC of tissue specific lipids plotted as a ratio of the Total TIC across voltages to determine which resulted in greater relative abundance of the lipids. (c, d) Panels are for MeOH

Figure 8

(a, d) The tissue specific lipid abundances are plotted as mentioned in Figure 5, shown from low to high m/z from top to bottom of each image. (b, e) the TIC of 133 assignable tissue specific lipids is plotted in blue and percent coverage of those lipids is in green. (c, f) In c and f the TIC of 133 assignable tissue specific lipids is plotted as a ratio of the whole TIC. A, b, c model the ACN gradients, and d, e, f model MeOH gradients