TITLE: Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) for Bulk Metabolomics
AUTHORS: Dusan Velickovic, Jessica Lukowski, Guanshi Zhang, Theodore Alexandrov, Chris Anderton, Kumar Sharma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mass spectrometry imaging is an exciting technologywhich enables a simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs,. In combination with histological methods, this technique provides information about the spatial distribution of molecules in various biological tissues. Particularly, MALDI-MS imaging increases the coverage of metabolites by using different matrices and ion modes. In coordination with in situ analysis of proteins, transcripts and epigenetic marks, the complementary spatial information on metabolites will establish metabolic pathways that are dominant and characteristic of disease states. We have recently developed and optimized a spatial metabolomics approach to image small molecules in human kidneys and biopsy sized material. With our combined expertise at UTHSA, PNNL and EMBL and recent advances, we have established methods for identifying metabolites in human kidneys, employed ultra-high mass resolution MS imaging for tissue analysis, and developed a bioinformatics resource (METASPACE) to annotate metabolites for anatomical localization and 3-D reconstruction. Our integrated technology can easily connect with other TIS sites to provide biochemical readouts of genes/proteins in specific tissue and cellular compartments.</div><div class = "text-block">Although METASPACE bioinformatics provides metabolite annotations of given reliability, there may be an inherent level of ambiguity due to the lack of untargeted MS/MS fragmentation in MALDI-MSI. Metabolite annotations from MALDI-MSI and METASPACE are structurally validated using orthogonal analytical techniques such as LC-MS/MS due to its high metabolome coverage, sensitivity, and throughput. The work flow for ‘bulk omics’ is shown in Figure 5.</div></div>

[STEPS]
?. [LC- MS/MS]
Vial is placed in autosampler of Waters H class LC.
?. [LC- MS/MS]
Inject   of sample onto a reversed phase Waters CSH column (3.0 mm x 150 mm x 1.7 μm particle size) with a 34 min gradient (mobile phase A: ACN/H2O (40:60) containing 10 mM ammonium acetate; mobile phase B: ACN/IPA (10:90) containing 10 mM ammonium acetate) at a flow rate of 250 μl/min to separate the lipid molecular species.
10 µl
The gradient should be made fresh for each run.
?. [LC- MS/MS]
Mass spectrometry is performed using a Thermo Velos-ETD Orbitrap mass spectrometer.
?. [LC- MS/MS]
The LC-MS/MS raw data files were analyzed using LIQUID and confident lipid identifications were determined as outlined in the main text.
?. [LC- MS/MS]
LC-MS data is also converted to mzML format and uploaded to LC-METASPACE
?. [LC- MS/MS]
Samples and are analyzed in both positive and negative ionization modes using HCD (higher-energy collision dissociation) and CID (collision-induced dissociation) to obtain high coverage of the lipidome
?. [LC- MS/MS]
Prior to LC analysis, dry sample in speed vac and reconstitute in   of 95:5 MeOH:Chl in an vial.
100 µl
?. [LC- MS/MS]
Add MilliQ water ( -  ) to biopsy tube containing remaining biopsy sample (material not sectioned) and lyse the remaining biopsy sample (material not sectioned) using a tissue lyser. Quantify the amount of tissue remaining by weight.
200 µl
300 µl
?. [LC- MS/MS]
Place the sample into a   or   Sorenson MµlTI™ SafeSeal™ Microcentrifuge Tube
1.7 ml
2 ml
These tubes have been shown to not leach polymers into the lipid layer from the chloroform.
?. [LC- MS/MS]
Add cold ( ) chloroform:methanol mix (prepared 2:1 v/v) to sample in 4:1 ratio over sample volume and vortex
-20 °C
For example add 400 µl of the 2:1 Chl:MeOH mixture to 80 µl of sample
?. [LC- MS/MS]
Carefully removed the upper aqueous metabolite layer until the interphase contracts without disturbing the protein disk, discard.
?. [LC- MS/MS]
The organic layer (containing lipids) is placed into the speed vac to dry.
?. [LC- MS/MS]
of 2:1 Chl:MeOH and cap (no septa) is store at   until ready for analysis (need extra solution volume for safe storage).
500 µl
-70 °C
?. [LC- MS/MS]
Vortex for  to
?. [LC- MS/MS]
Let stand on ice for
?. [LC- MS/MS]
Vortex for  to
?. [LC- MS/MS]
Centrifuge the sample at
Centrifuge: 12000 34, 4 10
?. [LC- MS/MS]
Carefully puncturing the protein interphase with a pipette tip, remove the organic lipid phase from the bottom of the tube (be sure to gently push out any protein or upper methanol phase that might have entered the pipette tip) into a conical bottom Waters autosampler vial.