Elastase Digests: New Ammunition for Shotgun Membrane Proteomics

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.Upon the introduction of modern mass spectrometric ionization techniques, such as MALDI (1) and ESI (2), extremely powerful and valuable tools were given to researchers for the identification and characterization of proteins. Nevertheless the intricate analysis of membrane subproteomes still represents one of the major challenges despite the amount of “success” reported in the literature. Until now, there was no general protocol available to address membrane proteomes as a whole, demonstrating their degree of difficulty and complexity.During the past years, two different strategies in proteome analysis have evolved: the more widespread bottom-up and top-down proteomics. The latter approach has been shown to provide access to the transmembranal regions of membrane proteins and has the power to characterize complete protein primary structures including labile covalent modifications (3, 4). Nevertheless there are limitations due to sample complexity, emphasizing the need for a liquid chromatographic separation on the protein level, which is comparable to LC peptide separation. Therefore, the bottom-up variant is the most commonly used method for the proteomics analysis of complex membrane samples. Improvements in the bottom-up work flow were achieved by adapting sample cleanup and prefractionation processes (5–10) and subsequently by the development of modified and optimized separation techniques. The two main work flows, the gel-based approach mainly carried out with 2D¹ SDS-PAGE (11, 12) and the shotgun identification of proteolytically digested protein mixtures and their multidimensional separation via liquid chromatography (13), had to be adapted. The separation of proteins via classical 2D SDS-PAGE is only possible up to a GRAVY score of ∼0.4 (14, 15). Despite lacking the separation power of the IEF-SDS-PAGE system, derived techniques like the doubled SDS- (16) and 16-benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE (17) represent an improvement for hydrophobic proteins. Advances in the nLC separation of hydrophobic peptides, e.g. the separation at elevated temperatures (18) and the use of LC-compatible detergents (19), yielded significant success. When combining both prominent separation techniques, the one-dimensional SDS-LC analysis proved to be more effective than 2D PAGE as well (20, 21).One of the remaining steps still offering room for improvements is the proteolytic procedure. The original multidimensional protein identification technique using trypsin has been successfully improved by the application of proteinase K under high pH conditions, yielding an increased number of membrane protein identifications (22). Further modification of this method by recleaving the isolated membranous parts with cyanogen bromide increased the accessibility to membrane-spanning peptides (18).Despite the suitability of proteinase K for the shotgun analysis of membrane proteomes, trypsin is the prevalent enzyme choice in most current proteomics approaches because of its very specific cleavage behavior. Calculations have implied that alternative proteases are preferentially suited for the analysis of membrane proteomes (23). Proteases other than trypsin have been utilized only to a small degree and mostly for the targeted analysis of protein complexes. Pepsin, for example, was used for the characterization of an aquaporin (24), and elastase and subtilisin were used as proteases in a triple digest approach of protein complexes and lens tissue (25). Further improvements in the accessibility of the membrane proteome have been shown for tryptic (26, 27) and tryptic/chymotryptic (28) digests by performing the proteolytic treatment in the presence of methanol.During membrane proteomics method development, purple membranes from Halobacterium sp. NRC-1, consisting to a large extent of the H⁺-ATPase bacteriorhodopsin, have widely been used as reference in a variety of cases. Full BR sequence coverage has been reported by several publications using different techniques (27, 29). Such exorbitant sequence coverage amounts are only achievable for the most abundant proteins within a complex mixture. The identification of less expressed proteins, which certainly are more in the researcher''s interest, occurs via lower peptide numbers (27).Generally the basic tryptic cleavage sites are predominantly located in the loops facing the cytoplasm and to a lesser extent in the extracellular or periplasmic loops but are very scarce within the transmembrane helix stretches. This mostly limits the potential tryptic fragments to loop components and larger peptides containing at least one TM helix. The use of another protease, preferentially cleaving after neutral aliphatic residues, should not succumb to this lack of cleavage sites.One of the previously mentioned proteases, porcine pancreatic elastase, was reported to possess potential cleavage specificity at the carboxyl-terminal side of small neutral amino acids (30). It has been used previously for the examination of single protein phosphorylations (31) but was described not to be suited for the cleavage of complex membrane-containing samples because of a supposedly limited activity when applied to them (22). Despite this finding, elastase has been successfully utilized in a mass spectrometric analysis as early as 1974 when it was regarded as an ideal protease for future mass spectrometric studies (32). At the same time, most of the experiments to characterize elastase by analyzing its S₁ pocket (for nomenclature, see Ref. 33) binding capabilities (34) as well as cleavage preferences of ester and protein substrates (35–39) were performed.Based on previous research concerning elastase, we set up an nLC-based membrane proteomics analysis. This large data set was used to characterize the protease behavior of elastase and the physicochemical properties of the detected peptides. The PM model was used to establish a method to analyze more complex Corynebacterium glutamicum membranes, which have been analyzed previously using different 2D techniques (7, 28). We demonstrated that a combination of elastase and methanol is particularly suitable for an nLC-based membrane proteome analysis and results in a significantly increased number of TM peptides. Additionally the promising PMF application of elastase digests is discussed.     

Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics

Protein identification by tandem mass spectrometry is based on the reliable processing of the acquired data. Unfortunately, the generation of a large number of poor quality spectra is commonly observed in LC-MS/MS, and the processing of these mostly noninformative spectra with its associated costs should be avoided. We present a continuous quality score that can be computed very quickly and that can be considered an approximation of the MASCOT score in case of a correct identification. This score can be used to reject low quality spectra prior to database identification, or to draw attention to those spectra that exhibit a (supposedly) high information content, but could not be identified. The proposed quality score can be calibrated automatically on site without the need for a manually generated training set. When this score is turned into a classifier and when features are used that are independent of the instrument, the proposed approach performs equally to previously published classifiers and feature sets and also gives insights into the behavior of the MASCOT score.     

An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.     

Genome-wide analysis of a land plant-specific acyl:coenzyme A synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella

The plant enzyme 4-coumarate:coenzyme A ligase (4CL) is part of a family of adenylate-forming enzymes present in all organisms. Analysis of genome sequences shows the presence of '4CL-like' enzymes in plants and other organisms, but their evolutionary relationships and functions remain largely unknown. 4CL and 4CL-like genes were identified by BLAST searches in Arabidopsis, Populus, rice, Physcomitrella, Chlamydomonas and microbial genomes. Evolutionary relationships were inferred by phylogenetic analysis of aligned amino acid sequences. Expression patterns of a conserved set of Arabidopsis and poplar 4CL-like acyl-CoA synthetase (ACS) genes were assayed. The conserved ACS genes form a land plant-specific class. Angiosperm ACS genes grouped into five clades, each of which contained representatives in three fully sequenced genomes. Expression analysis revealed conserved developmental and stress-induced expression patterns of Arabidopsis and poplar genes in some clades. Evolution of plant ACS enzymes occurred early in land plants. Differential gene expansion of angiosperm ACS clades has occurred in some lineages. Evolutionary and gene expression data, combined with in vitro and limited in vivo protein function data, suggest that angiosperm ACS enzymes play conserved roles in octadecanoid and fatty acid metabolism, and play roles in organ development, for example in anthers.     

BepiPred‐3.0: Improved B‐cell epitope prediction using protein language models

B‐cell epitope prediction tools are of great medical and commercial interest due to their practical applications in vaccine development and disease diagnostics. The introduction of protein language models (LMs), trained on unprecedented large datasets of protein sequences and structures, tap into a powerful numeric representation that can be exploited to accurately predict local and global protein structural features from amino acid sequences only. In this paper, we present BepiPred‐3.0, a sequence‐based epitope prediction tool that, by exploiting LM embeddings, greatly improves the prediction accuracy for both linear and conformational epitope prediction on several independent test sets. Furthermore, by carefully selecting additional input variables and epitope residue annotation strategy, performance was further improved, thus achieving unprecedented predictive power. Our tool can predict epitopes across hundreds of sequences in minutes. It is freely available as a web server and a standalone package at https://services.healthtech.dtu.dk/service.php?BepiPred-3.0 with a user‐friendly interface to navigate the results.     

Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain

Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3 × FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexes in addition to formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and their protein complexes.     

Major conformational change in the complex SF3b upon integration into the spliceosomal U11/U12 di-snRNP as revealed by electron cryomicroscopy

In some eukaryotes, a minor class of introns is removed by the U12-dependent spliceosome, which contains the small nuclear ribonucleoprotein (snRNP) heterodimer U11/U12. The U11/U12 di-snRNP forms a molecular bridge that functionally pairs the intron ends of the pre-mRNA. We have determined the three-dimensional (3D) structure of the human U11/U12 di-snRNP by single particle electron cryomicroscopy using angular reconstitution and random conical tilt. SF3b, a heteromeric protein complex functionally important for branch site recognition, was located in the U11/U12 di-snRNP by antibody labeling and by identification of structural domains of SF3b155, SF3b49, and p14. The conformation of SF3b bound to the U11/U12 di-snRNP differs from that of isolated SF3b: upon integration into the di-snRNP, SF3b rearranges into a more open form. The manner in which SF3b is integrated in the U11/U12 di-snRNP has important implications for branch site recognition. Furthermore, a putative model of the pre-mRNA binding to the U11/U12 di-snRNP is proposed.     

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.     

Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method

Background  

Many processes in molecular biology involve the recognition of short sequences of nucleic-or amino acids, such as the binding of immunogenic peptides to major histocompatibility complex (MHC) molecules. From experimental data, a model of the sequence specificity of these processes can be constructed, such as a sequence motif, a scoring matrix or an artificial neural network. The purpose of these models is two-fold. First, they can provide a summary of experimental results, allowing for a deeper understanding of the mechanisms involved in sequence recognition. Second, such models can be used to predict the experimental outcome for yet untested sequences. In the past we reported the development of a method to generate such models called the Stabilized Matrix Method (SMM). This method has been successfully applied to predicting peptide binding to MHC molecules, peptide transport by the transporter associated with antigen presentation (TAP) and proteasomal cleavage of protein sequences.     

The design and implementation of the immune epitope database and analysis resource

Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, The Immune Epitope Database and Analysis Resource. We describe how we plan to capture, structure, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide.