Co-fractionation mass spectrometry (CF-MS) uses biochemical fractionation to isolate and characterize macromolecular complexes from cellular lysates without the need for affinity tagging or capture. In recent years, this has emerged as a powerful technique for elucidating global protein-protein interaction networks in a wide variety of biospecimens. This review highlights the latest advancements in CF-MS experimental workflows including machine learning-guided analyses, for uncovering dynamic and high-resolution protein interaction landscapes with enhanced sensitivity, accuracy and throughput, enabling better biophysical characterization of endogenous protein complexes. By addressing challenges and emergent opportunities in the field, this review underscores the transformative potential of CF-MS in advancing our understanding of functional protein interaction networks in health and disease.

Proteomics technologies are continually advancing, providing opportunities to develop stronger and more robust protein interaction networks (PINs). In part, this is due to the ever-growing number of high-throughput proteomics methods that are available. This review discusses how data-independent acquisition (DIA) and co-fractionation mass spectrometry (CF-MS) can be integrated to enhance interactome mapping abilities. Furthermore, integrating these two techniques can improve data quality and network generation through extended protein coverage, less missing data, and reduced noise. CF-DIA-MS shows promise in expanding our knowledge of interactomes, notably for non-model organisms (NMOs). CF-MS is a valuable technique on its own, but upon the integration of DIA, the potential to develop robust PINs increases, offering a unique approach for researchers to gain an in-depth understanding into the dynamics of numerous biological processes.

Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.