A Membrane‐Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross‐Linking Reagent to Advance In Vivo Cross‐Linking Mass Spectrometry

Abstract

Cross-linking mass spectrometry (XL-MS) is an attractive method for the proteome-wide characterization of protein structures and interactions. Currently, the depth of in vivo XL-MS studies is lagging behind the established applications to cell lysates, because cross-linking reagents that can penetrate intact cells and strategies to enrich cross-linked peptides lack efficiency. To tackle these limitations, we have developed a phosphonate-containing cross-linker, tBu-PhoX, that efficiently permeates various biological membranes and can be robustly enriched using routine immobilized metal ion affinity chromatography. We have established a tBu-PhoXbased in vivo XL-MS approach that enables cross-links in intact human cells to be identified in high numbers with substantially reduced analysis time. Collectively, the developed cross-linker and XL-MS approach pave the way for the comprehensive XL-MS characterization of living systems.

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

“…Besides, proper crosslinking time should be adopted to reduce the disturbance of the crosslinking reaction to the cellular native state . Recently, the well-established tBu-PhoX-based CXMS pipeline showed great potential for in vivo crosslinking in 30 min without obvious cellular disturbance . Therefore, how to efficiently capture the dynamics of protein complexes with finer temporal resolution and good biocompatibility to living cells by a crosslinker with cell membrane permeability and fast crosslinking fixing was another attractive and important query.…”

“…As a response, many strategies have been developed to enhance the enrichment of crosslinked peptides, including chromatographic techniques such as strong cation exchange or size exclusion chromatography or directly utilization of an enrichable trifunctional crosslinker. Among these strategies, the development of an enrichable crosslinker is the most promising strategy, such as BDP-NHP, NNP9, Alkyne-A-DSBSO, tBu-PhoX or Phox, Leiker, and so forth, exhibiting a high enrichment selectivity in complex peptide mixtures. More significantly, a smaller enrichment handle of azide or alkyne that allows derivatization to introduce a biotin by biorthogonal click chemistry ,,− followed by highly specific biotin-streptavidin enrichment might be a recommended choice for crosslinkers to alleviate the strong hydrophobicity and steric hindrance of the biotin.…”

Real-Time Library Search Increases Cross-Link Identification Depth across All Levels of Sample Complexity

“…Cross-linked and mono-linked peptides were enriched as described before. 19 LC-MS Data Acquisition. Samples were separated by reverse-phase HPLC using a Thermo Scientific EASY-nLC 1200 system connected to an EASY-Spray PepMap RSLC C18 column [0.075 mm × 250 mm, 2 μm particle size, 100 Å pore size (Thermo Fisher Scientific)] at a 300 nL/min flow rate.…”

“…Cross-linked peptides were desalted using Sep-Pak C8 cartridges (Waters) and dried. Cross-linked and mono-linked peptides were enriched as described before …”

“…We recently described a sample processing workflow, in particular a two-step immobilized metal affinity chromatography (IMAC) enrichment strategy for interactome profiling of intact cells based on the enrichable cross-linker tert -butyl-PhoX (tBu-PhoX) . In this study, we further augment the tBu-PhoX XL-MS pipeline from the data acquisition perspective by integrating RTLS to advance cross-link detection.…”