Table 2.

Summary of the advantages and disadvantages of local proteomics approaches.

Approach	Advantages	Disadvantages
Laser capture microdissection (LCM)	• Ability to image cell type and structure and acquire cell count • A wide range of tissues can be used: unfixed frozen postmortem brain tissue, formalin-fixed paraffin embedded brain tissue, hematoxylin & eosin stained or immunostained tissues • One tissue section can be dissected several times for different regions • Dissection does not disturb cells’ molecular state • Area size of 1.5 mm2 is sufficient for MS	• Tissue drying during dissection • Dissection process can be time-consuming: 5 min to 8 hrs depending on the size, cell type, and number of areas or cells to be collected • Inability to confidently exclude cells that are not of interest • Protocol not optimized for smaller, non-neuronal cell types or single cells • Lower proteome coverage compared to bulk brain proteomics
Magnetic-activated cell sorting (MACS)	• High throughput • High purity • Selective and rapid method • Enrichment can be scaled up or down to desired yield	• Contamination by acellular debris or unbound cells • Immunomagnetic beads might cause mechanical shear • Sequential isolations significantly reduce yield • Cannot isolate intact neurons
Fluorescence-activated cell sorting (FACS)	• High sensitivity, throughput, and purity • Isolate multiple cell types simultaneously based on immunopheno type alone • Sort complex cell types with multiple markers • Separate cells based on cell size, density, and morphology, cell cycle status, intracellular cytokine expression, and metabolic profile • Capture immunophenotyping data for 12 surface epitopes • Minimum of 12,000 cells are sufficient for MS	• Long isolation procedure (3+ hrs) • Shear stress from the FACS instrument • Slow sorting process – depends on number of cell populations that need to be collect • Recovery is 50–70% on most sorters, need a high number of cells at the beginning • Fluorophore spillover into non-specific channels between cells with closely related immune phenotypes • Cannot isolate intact neurons
Bio-orthogonal non-canonical amino acid tagging (BONCAT)	• Identification of low abundance, low copy number newly synthesized proteins with higher magnitude • Click chemistry procedure is modular and relatively simple • Lineage tracing of proteins • Established transgenic mouse line under the Cre/Lox system	• Cost of Anl and special diet for mouse studies • Obtaining a good signal-to-noise ratio between endogenously biotinylated proteins and biotin clicked Anl-tagged proteins • Depth of proteome is lower than traditional proteomics • Reduced labeling efficiency due to competition between endogenous MetRS and MetRS*
Proximity labeling (BioID, APEX, TurboID)	• Rapid kinetics of biotinylation without click chemistry • Detect weak or transient protein interactions as well as soluble and insoluble proteins • Ready bioavailability of biotin in the brain after peripheral administration • Acquire a more global proteome unlike the nascent proteins in BONCAT	• Noise introduced by endogenous biotinylation • APEX approach is limited to in vitro experiments since biotin-phenol is toxic • TurboID can sequester endogenous biotin and cause toxicity • Saturation of proximal labeling sides with prolonged biotin supplementation • Lack of mouse models for BioID and TurboID approaches