Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics

Abstract

This article presents the strategies underlying the automated identification and quantification of individual lipid molecular species through array analysis of multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) data, which are acquired directly from lipid extracts after direct infusion and intrasource separation. The automated analyses of individual lipid molecular species in the program employ a strategy in which MDMS-SL data from building block analyses using precursor ion scans, neutral loss scans, or both are used to identify individual molecular species, followed by quantitation. Through this strategy, the program screens and identifies species in a high-throughput fashion from a built-in database of over 36,000 potential lipid molecular species constructed employing known building blocks. The program then uses a two-step procedure for quantitation of the identified species possessing a linear dynamic range over 3 orders of magnitude and reverifies the results when necessary through redundant quantification of multidimensional mass spectra. This program is designed to be easily adaptable for other shotgun lipidomics approaches that are currently used for mass spectrometric analysis of lipids. Accordingly, the development of this program should greatly accelerate high-throughput analysis of lipids using MDMS-based shotgun lipidomics.

Figure 1

Total ion current chromatogram of stepwise acquisition of MS and tandem MS spectra for identification and quantitation of phosphatidylinositol molecular species in the lipid extracts of mouse myocardium. The lipid extract from mouse myocardium was analyzed in the negative-ion mode after infusion of the diluted lipid extract directly with a nanomate device. The stepwise acquisition of MS and tandem MS spectra as indicated (Panel A) was conducted using a sequential and customized program operating under Xcalibur software. Each segment of individual PIS was taken for 2 min in the profile mode. Panel B shows an example of the PIS averaged from all of scans acquired in the segment corresponding to PIS329 (i.e., 22:5 FA).

Figure 2

A representative two-dimensional mass spectrometric analysis of phosphatidylinositol molecular species present in the lipid extracts of mouse myocardium. Each MS or MS/MS scan of the 2D ESI mass spectrum was acquired by sequentially programmed, customized scans operating under Xcalibur software. All scans were displayed after being normalized to the base peak in each individual scan. “IS” denotes internal standard (at m/z 871.5); “GPI” stands for glycerophosphoinositol.

Scheme 1

The general structures of glycerophospholipid, glycerolipid, and sphingolipid with building blocks.

Scheme 2

A schematic of the workflow used in AMDMS-SL to identify individual molecular species in a lipid class of interest.

Scheme 3

A schematic of the workflow used in AMDMS-SL to quantify individual molecular species in a lipid class of interest. The I_MS and I_MS/MS are the normalized peak intensities in the full MS scan and the MS/MS scan which is specific to the lipid class of interest, respectively. The c_u and c_i are the content of individual species in the list and the selected internal standard, respectively, and I_u and I_i are the peak intensities of the species and the selected internal standard, respectively, after ¹³C de-isotoping.