Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

Saturated mutagenesis of ketoisovalerate decarboxylase V461 enabled specific synthesis of 1-pentanol via the ketoacid elongation cycle.

Iterative ketoacid elongation has been an essential tool in engineering artificial metabolism, in particular the synthetic alcohols. However, precise control of product specificity is still greatly challenged by the substrate promiscuity of the ketoacid decarboxylase, which unselectively hijacks ketoacid intermediates from the elongation cycle along with the target ketoacid. In this work, preferential tuning of the Lactococcus lactis ketoisovalerate decarboxylase (Kivd) specificity toward 1-pentanol synthesis was achieved via saturated mutagenesis of the key residue V461 followed by screening of the resulting alcohol spectrum. Substitution of V461 with the small and polar amino acid glycine or serine significantly improved the Kivd selectivity toward the 1-pentanol precursor 2-ketocaproate by lowering its catalytic efficiency for the upstream ketoacid 2-ketobutyrate and 2-ketovalerate. Conversely, replacing V461 with bulky or charged side chains displayed severely adverse effect. Increasing supply of the iterative addition unit acetyl-CoA by acetate feeding further drove 2-ketoacid flux into the elongation cycle and enhanced 1-pentanol productivity. The Kivd V461G variant enabled a 1-pentanol production specificity around 90% of the total alcohol content with or without oleyl alcohol extraction. This work adds insight to the selectivity of Kivd active site.