Investigation of the Effects of Labware Contamination on Mass Spectrometry-Based Human Serum Lipidome Analysis

Abstract

Polypropylene microcentrifuge tubes (MCTs) are increasingly used in lipidome sample preparation. In the absence of a comprehensive study evaluating ramifications of plasticware utilization in mass spectrometry-based lipidomic analyses, we conducted a systematic analysis to elucidate potential negative effects ascribable to labware contamination in serum lipidomics. During serum lipid extractions, tested glassware introduced 24 labware contaminants. In contrast, Eppendorf polypropylene MCTs contributed 485 contaminant features, many of which could be erroneously putatively identified as lipids via their m/z values. Eppendorf MCTs contamination engendered severe ionsuppression of 40 low abundance serum lipids, while generating mild to modest lipid ion-suppression across a multitude of higher abundance coeluting lipids. Less compatible polypropylene MCTs from an alternative manufacturer introduced a staggering 2,949 contaminant m/z values, severely affecting 75 coeluting serum lipids and causing more frequent and pronounced ion-suppression instances. Furthermore, by performing serum extractions with varied initial volumes, it was ascertained that labware-induced lipid ion-suppression is a dynamic phenomenon, contingent on both lipid and labware contaminant concentrations where low-abundance lipids are disproportionately impacted by coelutes of suppressive contaminants. In addition to lipid ion-suppression, the identification and quantification of 7 fatty acid endogenous serum lipids were compromised by the leaching of structurally identical surfactants from MCTs. MCTs artificially introduced 10 additional primary amides extraneous to serum samples. Utmost caution is imperative in interpreting data concerning primary amides and fatty acids when employing plastic labware. Through this investigation, we aspire to elevate awareness regarding the pernicious impact of labware contamination on lipidome analysis.

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

“…The assignment of low abundant signals (0.73% of n -7 signal intensity) consistent with oleic acid (FA 18:1 n- 9, cis ) in the FA 18:1 n- 7, cis OzID data (Figure A) are very likely the result of the former being a common background contaminant in ESI-MS, with a recent study highlighting that a variety of background lipids from labware and solvent contamination may interfere with quantitation at very low concentrations . Conversely, the considerably lower abundance of n- 7 signals (only 0.14% of n -9 signal intensity, Figure C) in the reference data from FA 18:1 n- 9, cis suggests that cross contamination between samples was negligible and instead suggests that these signals result from ion chemistry within the mass spectrometer.…”

“…The observation of OzID transitions outside those expected for the site of unsaturation in the reference fatty acids indicates one or more of the following: (i) the reference standards include trace amounts of alternate fatty acid isomers; (ii) ionization, mass-selection or ion ejection of the [M–H+2Li] + cations promotes some fragmentation or isomerization and/or; (iii) the ion activation modality itself promotes competing or secondary chemistries giving rise to false positives. Considering each of these in turn: The assignment of low abundant signals (0.73% of n -7 signal intensity) consistent with oleic acid (FA 18:1 n- 9, cis ) in the FA 18:1 n- 7, cis OzID data (Figure A) are very likely the result of the former being a common background contaminant in ESI-MS, with a recent study highlighting that a variety of background lipids from labware and solvent contamination may interfere with quantitation at very low concentrations . Conversely, the considerably lower abundance of n- 7 signals (only 0.14% of n -9 signal intensity, Figure C) in the reference data from FA 18:1 n- 9, cis suggests that cross contamination between samples was negligible and instead suggests that these signals result from ion chemistry within the mass spectrometer. [M–H+2Li] + ions formed from fatty acids have previously been utilized to promote charge-remote fragmentation; − a process of unimolecular dissociation that cleaves carbon–carbon bonds and produces terminal alkene product ions that could potentially undergo reaction with ozone.…”

Trace elements increase reproducibility of microbial growth

“…However, a concentration of zero is an unattainable ideal. Possible sources of contamination include impurities in ingredients [13][14][15] and labware 16 , production and cleaning residues on labware 17 , and aerosols 18 . Since trace elements already take effect at very low levels, we expect such contamination to cause considerable experimental variability (Fig.…”