Table 1.

A table listing the pros and cons of   MS-based methodologies for identifying and quantifying PPIs

Methods	Pros	Cons
AP-MS	Co-immunoprecipitation can be performed without tagged baits expressed at physiological levels to identify endogenous PPIs	Co-IP with untagged baits is limited by the availability of antibodies, and the low expression levels of baits
	Epitope tagging provides an alternative for purifying proteins lacking suitable antibodies	Epitope tags may interfere with the functions and solubility of the baits
	Transient transfection of tagged baits enhance their expression, thus improving the efficiency and throughput of the pulldowns	Ectopic expression of tagged baits may promote misfolding and mislocalization of the baits, promoting background contamination and spurious interactions
PDB-MS	Allows detection of PPIs among both soluble and membrane proteins, as well as enriching for PPIs that are transient, weak, low abundance or have high turnover [11, 28, 29]	May react with biotin-phenol and H2O2 to produce reactive radicals resulting in cellular toxicity (APEX) [30]
	Avoids post-lysis artefacts [11]	The accessibility and labelling efficiency of the biotinylating enzyme are locality-dependent, as its orientation and topology within the protein complex may impede its performance
	The affinity of biotin to streptavidin is robust yet reversible. Hence, highly stringent conditions for sample denaturing, solubilization, capture, wash and extraction of biotinylated proteins can be employed to maximize the recovery of hydrophobic proteins while minimizing nonspecific background contaminants	The high affinity of the streptavidin–biotin interaction may hinder the recovery of highly biotinylated proteins. PDB-MS suffers from false positives in the forms of high-abundance background proteins or artefacts from endogenous biotinylation
		The labelling time for different enzyme varies from 1 min to 24 h [12, 13, 16]
XL-MS	Crosslinking reagents can covalently connect two or more non-covalently interacting proteins, regardless of the duration and strength of the interaction. As such, even transient and weak PPIs can be preserved [45, 46]	The low efficiency (~ 1–5%) of crosslinking reagents, which often results in marginal crosslinks, where only the top 20–30% of proteins are detected
	When used in combination with X-ray crystallography, CryoEM, NMR and native MS, the spatial constraint data from XL-MS can guide molecular modelling, construct a connectivity map for determining subunit topology, and map the dynamic behaviour of the protein complex [49–51 ]	The crosslinking reaction time may be relatively long (~ 30 min). Excessively long reaction time may result in large, crosslinked protein aggregates
	To expand the number and coverage of crosslinks, alternative modes of crosslinking can be employed, such as carboxyl-targeting reagents [40–42]	A crosslinker covalently links two linear peptides, giving rise to a hybrid dipeptide that can dramatically expand the search space during spectra matching, giving rise to the 'n-square problem' [68, 69]
Co-Frac-MS	CoFrac-MS has high throughput, and it provides global identification and quantification of native protein complexes in one setting	False positives constitute a significant problem in the form of chance co-elution
	It can be operated without genetic manipulation and overexpression, thereby inferring endogenous, physiologically relevant interactome [3]
	CoFrac-MS combined with quantitative proteomics can delineate the relative distribution of a protein in multiple co-elution features. Thus, the stoichiometries and dynamics of a target protein within different co-isolated complexes can be simultaneously elucidated [85]
TPCA	TPCA permits system-wide profiling of protein complex dynamics, and it requires neither antibodies nor epitope tagging [87]	The current version of TPCA is limited to studying the dynamics of known or predicted protein complexes across cellular state and physiological conditions. Need to incorporate existing interaction data with graph/network clustering algorithms to identify novel protein complexes [87]
	Little preparation time is required. It allows most of the study of protein complexes in situ and in vivo
	TPCA profiling can be rapidly deployed to unravel the assembly state of protein complexes across cellular state, cell type, tissue and physiological conditions to provide insight into their functions in normal and diseased cells