Reliable Identification of Cross-Linked Products in Protein Interaction Studies by ¹³C-Labeled p-Benzoylphenylalanine

Abstract

We describe the use of the (13)C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a (13)C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

Photocross-Linked Peptide–Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes

“…Therefore, even if MALDI-TOF/TOF cannot be easily automated in an on-line workflow (nanoLC coupling and automatic acquisition) and thus would not be suitable for high throughput proteomic/ interactomic analysis, it can provide occasional and very valuable information in the case of cross-linking-based structural studies. However, low-energy CID fragmentation of the C-C bond has been recently reported in a similar study concerning the interaction between the photoreactive peptide M13 and its partner protein CaM using the para-benzoyl phenylalanine as the photoprobe [24]. In this case the fragmentation of the newly formed C-C bond is believed to be specific of cross-linked products with N-terminal and oxidized methionines and results from consecutive fragmentation.…”

A presequence-binding groove in Tom70 supports import of Mdl1 into mitochondria

“…Tom20 and Tom22 recognize the N-terminal targeting 53 signal (presequence) of precursor proteins destined for the mitochondrial 54 matrix. The subsequent transport of these precursors through the TOM 55 complex is then mediated by the small Tom5 and the channel-forming 56 Tom40 [8 -11]. The presequence is characterized by its ability to form an 57 amphipathic α-helix.…”

“…Fragment ions resulting from cleavage of the cross-link bond are labeled with the respective tryptic peptide sequence. Asterisks indicate characteristic signals that are frequently observed after fragmentation of cross-linked methionine side chains [11,33,56]. C) Structural model of yeast Tom70 (PDB ID: 2GW1; residues 94-607) as cartoon (left).…”

In planta chemical cross‐linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

“…Developing an in vivo proteomic approach has become urgently needed to better understand the biological processes under complex cellular environments. Recently, chemical cross-linking in combination with MS (CX-MS) has emerged as a popular alternative MS approach to the investigation of model protein folding, protein structure, and PPIs [9][10][11][12][13][14][15][16][17][18][19]. Among these emerging approaches, a nonspecific cross-linking reagent, such as formaldehyde, is used as the simplest cross-linker [15].…”