Ltq velos ion trap mass spectrometer

For the global search of lipid molecules from each sample group (8 groups in total), a model 1200 capillary pump system equipped with an autosampler (Agilent Technologies, Palo Alto, CA, USA) coupled to an LTQ Velos ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) was utilized with an analytical column. For targeted quantitation of identified lipids from global search, the SRM-based quantitation method was employed with UPLC–ESI–MS/MS using a nanoACQUITY UPLC system (Waters, Milford, MA, USA) equipped with an autosampler and a TSQ Vantage triple-stage quadrupole MS system (Thermo Scientific). The list of product ions used for SRM along with each collision energy value are listed in Supplementary Table S5. Details are found in Supplementary Information online.

Excised Colloidal Blue-stained gel bands were cut into approximately 1 mm³ pieces and then subjected to a modified in-gel trypsin digestion procedure as described previously [39 (link)]. Gel pieces were washed, dehydrated with acetonitrile and then rehydrated with 50 mM NH₄HCO₃ containing 12.5 ng/μl modified sequencing-grade trypsin (Promega, Madison, WI) for 45 min at 4 °C. Peptides were extracted by removing the NH₄HCO₃ solution, followed by one wash with a solution containing 50 % acetonitrile and 1 % formic acid, dried and stored at 4 °C. On the day of analysis, samples were reconstituted in HPLC solvent A (2.5 % acetonitrile, 0.1 % formic acid) and loaded onto a nano-scale reverse-phase HPLC capillary column via a Famos auto sampler (LC Packings, San Francisco, CA). Peptides were eluted with the help of increasing concentrations of solvent B (97.5 % acetonitrile, 0.1 % formic acid), subjected to electrospray ionization and then entered into an LTQ Velos ion-trap mass spectrometer (Thermo Fisher, San Jose, CA). Peptides were detected, isolated, and fragmented to generate a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program Sequest (ThermoFisher, San Jose, CA).