LipidSearch Troubleshooting

Depending on acquisition parameters 5,000 to 10,000 or more MS² spectra may obtained in a Top N experiment during a 30 min run. The number of sum composition lipids detected and integrated corresponds to the number of lipid groups. The number of lipid ions corresponds to the number of lipid species identified from the MS² spectra.

At the MS-MS level this is not possible. However, lipid retention time can be modeled with lipid standards to determine the likely candidates. Right now, double bond position needs to be determined by a separate method such as hydrolysis followed by GC/MS, by ion-molecule reactions (ozone) or UVPD (photodissociation). LipidSearch4.2_FAQ_7.16.2018.docx

The method of identification is the same from dd-MS² data. However, since several overlapping precursor ions may be co-isolated, infusion typically results in a lower number of lipid species identified.

LipidSearch is looking for fragments of the intact lipid precursor ions which is best achieved using ESI. LipidSearch supports identification of lipids that give intact molecular adduct ions. For example, negative ion APCI of fatty acids gives Cl adducts, and these adducts are included in the LipidSearch database.

Full or micro flow rates are typically used for lipid separations, whereas nano-ESI is preferred for infusion. One practical reason is that lipid carryover can be much worse at nanoliter/min flow rates.

The mass tolerance for the precursor ion is ±5 ppm. In TOP N dd-MS² experiments the accurate mass of the precursor ion triggering the MS² is stored in the spectrum file header.

TraceFinder requires chromatographic peaks for integration, but LipidSearch does not. LipidSearch can provide some limited relative quantitation from infusion data. TraceFinder is required for targeted LC-MS/MS methods.

As long as the experiment is full scan and dd-MS² the spectra can either be summed together (Infusion) or treated as a chromatogram (LC-MS) and the data processed by LipidSearch.

Identification in direct infusion is the same for dd-MS² spectra. However, mixtures of different precursor ions or isomers co-isolated at the same m/z gives rise to mixture MS² spectra. As a result, ID may be less accurate and the number of species identified is typically lower. We don’t recommend LipidSearch for the infusion analysis of complex lipid mixtures.

LipidSearch only includes basic statistic calculations including mean, standard deviation, % RSD and p-values. To do more sophisticated statistics, use the export function to create a data table in Microsoft Excel format.

Triacylglycerol species form NH4 adducts under ESI conditions and species with varying numbers of double bonds typically have different response factors. One way to compensate for this is to model using lipid standards and then apply an average response factor for TG species with varying numbers of double bonds. See reference: Han, X. and R.W. Gross, Anal. Biochem. 2001, 295, 88-100.

Identification in direct infusion is the same for dd-MS² spectra. However, mixtures of different precursor ions or isomers co-isolated at the same m/z gives rise to mixture MS² spectra. As a result, ID may be less accurate and the number of species identified is typically lower. We don’t recommend LipidSearch for the infusion analysis of complex lipid mixtures.