Session MOG. There are 4 abstracts in this session.

Session: Structural and Chemical Proteomics, time: 4:30 - 4:55 pm

Cross-linking Mass Spectrometry Strategies to Define Protein-Protein Interactions

Lan Huang
UC Irvine, Irvine, CA

Protein-protein interactions (PPIs) are fundamental to the formation of protein complexes and crucial for regulating various cellular activities. Cross-linking mass spectrometry (XL-MS) have become an emergent technology for mapping PPIs at the systems-level and elucidating architectures of large protein complexes. In comparison to standard structural methods, XL-MS approaches offer distinct advantages due to speed, accuracy, sensitivity and versatility, especially for the study of heterogeneous and dynamic protein complexes. Despite its great potential, XL-MS analysis remains challenging due to the difficulty in effective detection and identification of cross-linked peptides. Here, we will describe the development of sulfoxide-containing MS-cleavable cross-linking reagents (e.g. disuccinimidyl sulfoxide (DSSO)) for advancing MS analysis and identification of cross-linked peptides1,2. In addition, their applications in defining PPIs and structural dynamics of protein complexes will be presented. The analytical methods presented here can be directly applied to study PPIs in other biological systems. Ref 1. Kao, A. et, al. MCP, 2011; 2. Yu, C. et al, Anal. Chem. 2018.

Tips and Tricks (if submitted):

Session: Structural and Chemical Proteomics, time: 4:55 - 5:20 pm

In-Cell Protein Footprinting Coupled with Mass Spectrometry for Proteome-Wide Structural Biology

Lisa Jones
University of Maryland, Baltimore, MD

In recent years, protein footprinting coupled with mass spectrometry has been used to identify protein-protein interaction sites and regions of conformational change through modification of solvent accessible sites in proteins. The footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to modify solvent accessible sites in proteins. To date, FPOP has been used in vitro on relatively pure protein systems. We have further extended the FPOP method for both in cell and in vivo analysis of proteins. This will allow for study of proteins in their native cellular environment and be especially useful for the study of membrane proteins which can be difficult to purify for in vitro studies. We have designed and built a single cell flow system to enable uniform access of cells to the laser. Results demonstrate that in cell FPOP (IC-FPOP) can oxidatively modify over 1300 proteins in various cellular compartments. Owing to the high number of proteins that can be modified by IC-FPOP, we can use the method for proteome-wide structural biology. By comparing breast cancer cells treated with vehicle or with the anti-cancer drug Gleevec, we can identify on and off targets of the drug. We have further extended the method for in vivo analysis in C. elegans. We have demonstrated that with using an optimized flow system and a higher laser frequency we can modify over 500 proteins in various organisms within the worms. This demonstrates that both in-cell and in vivo FPOP can be used to study proteins in their native cellular environment.

Tips and Tricks (if submitted):

Session: Structural and Chemical Proteomics, time: 5:20 - 5:35 pm

Understanding the role of H2A proteolysis during stem cell differentiation

Mariel Coradin; Kelly Karch; Simone Sidoli; Benjamin A Garcia
University of Pennsylvania School of Medicine, Philadelphia, PA

Histone proteolysis is a poorly understood process by which the N-terminal tails get irreversible cleaved (clipped). This process has been described in cellular senescence, inflammation and stem cell differentiation, where its role remains unclear. In this study we combined Top-down MS, transcriptomics, and structural proteomics to interrogate the functional role of clipped H2A (cH2A) during stem cell differentiation and assess its consequences on nucleosome stability. Our data showed that H2A is cleaved during mouse embryonic stem cells (mESCs) differentiation by the lysosomal protease Cathepsin L. Using Top-Down MS, we were able to map the major cleavages sites to be at L23 and G44 (cH2A).  Cells treated with Cathepsin L inhibitors showed lower levels of cH2A, indicating that Cathepsin L also serves as H2A protease in vivo. Using RNA-sequencing, we found that inhibition of this enzyme leads to upregulation of genes involved in endoderm formation. We also assessed the modification landscape of H2A in mESCs. Using bottom-up proteomics, we found that the N-terminal tail is dynamically modified during this process. Similar to H3 and H4, acetylation levels of H2A increase in early stages of development, and as lineage commitment continues, acetylation levels drop significantly. We have also identified members of SWI/SNF chromatin remodeler complex (BRG1, SMARCC2) as binding acetylated H2A. Proteins in this complex are essential in embryonic cell self-renewal, highlighting a potential role of acetylated H2A in early development.  Finally, we sought to probe the structural consequences of cH2A, which lacks the alpha-1 helix. Using in vitro reconstituted dimers, we compared the hydrogen-deuterium exchange rate of full-length H2A and cH2A containing complexes. Our findings reveal that cH2A/H2B dimers are less stable than full-length H2A dimers. Taken together, our data suggest that histone proteolysis could be a novel mechanism for nucleosome eviction during mammalian development.   

Tips and Tricks (if submitted):

Session: Structural and Chemical Proteomics, time: 5:35 - 5:50 pm

Towards protein structure determination in living cells using a new translationally incorporated crosslinker that improves mass spectrometric detection

Bjorn-Erik Wulff; Josh Elias; Pehr Harbury
Stanford University, Stanford, CA

Atomic-resolution protein structures have been critical for biological discovery of life's molecular basis. A major current limitation, however, is that protein structures cannot be determined inside cells, where proteins exist in their native environment. This deficiency particularly impacts membrane proteins, cytoskeletal proteins, protein super-complexes, intrinsically disordered proteins and proteins that undergo phase separation in vivo. High‑throughput crosslinking mass spectrometry (XLMS) is an emerging technology with potential to provide the missing information. Analogously to NMR structure determination, XLMS can read out large numbers of amino acid proximity constraints that define the three‑dimensional protein fold. Importantly, the crosslinks must be short and distributed throughout the protein core, necessitating translational incorporation of a crosslinker that is typically light-activatable. However, such crosslinkers are devilishly difficult to detect by mass spectrometry.

We synthesized and tested a new family of ultra-short and light-activatable crosslinkers. We show that these can be translationally incorporated in yeast, and we present a path to incorporating them in other organisms. Two advances improve detection of the resulting crosslinks. First, we created a technique that depletes a trypsinate of peptides with only a single N-terminus. This removes the excess of linear peptides and leaves concentrated crosslinked peptides, which have two N-termini. This technique is agnostic to the details of the crosslink and the protease used. Second, our amino acid analogs incorporate a thioether bond that can be broken by very gentle CID that does not affect other bonds. It separates the crosslinked peptides from each other and any background species at the MS2 level and sequences them individually at the MS3 level. The 'mass scars' left by the crosslinker identify the individual amino acids that formed the crosslink. We show that custom control software for the mass spectrometer, which searches for and exploits this behavior, achieves speed and accuracy with great sensitivity.

Tips and Tricks (if submitted):