Optimization of Electrospray Ionization Source Parameters for Lipidomics To Reduce Misannotation of In-Source Fragments as Precursor Ions

Lipidomics
requires the accurate annotation of lipids in complex
samples to enable determination of their biological relevance. We
demonstrate that unintentional in-source fragmentation (ISF, common
in lipidomics) generates ions that have identical masses to other
lipids. Lysophosphatidylcholines (LPC), for example, generate in-source
fragments with the same mass as free fatty acids and lysophosphatidylethanolamines
(LPE). The misannotation of in-source fragments as true lipids is
particularly insidious in complex matrixes since most masses are initially
unannotated and comprehensive lipid standards are unavailable. Indeed,
we show such LPE/LPC misannotations are incorporated in the data submitted
to the National Institute of Standards and Technology (NIST) interlaboratory
comparison exercise. Computer simulations exhaustively identified
potential misannotations. The selection of in-source fragments of
highly abundant lipids as features, instead of the correct recognition
of trace lipids, can potentially lead to (i) missing the biologically
relevant lipids (i.e., a false negative) and/or (ii) incorrect assignation
of a phenotype to an incorrect lipid (i.e., false positive). When
ISF is not eliminated in the negative ion mode, ∼40% of the
100 most abundant masses corresponding to unique phospholipids measured
in plasma were artifacts from ISF. We show that chromatographic separation
and ion intensity considerations assist in distinguishing precursor
ions from in-source fragments, suggesting ISF may be especially problematic
when complex samples are analyzed via shotgun lipidomics. We also
conduct a systematic evaluation of electrospray ionization (ESI) source
parameters on an Exactive equipped with a heated electrospray ionization
(HESI-II) source with the objective of obtaining uniformly appropriate
source conditions for a wide range of lipids, while, at the same time,
reducing in-source fragmentation.