Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation

Abstract

Transmembrane proteins (TMP) play a crucial role in several physiological processes. Despite their importance and diversity, only a few TMP structures have been determined by high-resolution protein structure characterization methods so far. Due to the low number of determined TMP structures, the parallel development of various bioinformatics and experimental methods was necessary for their topological characterization. The combination of these methods is a powerful approach in the determination of TMP topology as in the Constrained Consensus TOPology prediction. To support the prediction, we previously developed a high-throughput topology characterization method based on primary amino group-labelling that is still limited in identifying all TMPs and their extracellular segments on the surface of a particular cell type. In order to generate more topology information, a new step, a partial proteolysis of the cell surface has been introduced to our method. This step results in new primary amino groups in the proteins that can be biotinylated with a membrane-impermeable agent while the cells still remain intact. Pre-digestion also promotes the emergence of modified peptides that are more suitable for MS/MS analysis. The modified sites can be utilized as extracellular constraints in topology predictions and may contribute to the refined topology of these proteins.

Putative linear motifs mediate the trafficking to apical and basolateral membranes

“…Although some of these proteins are available in high-throughput sets [31], each of these proteins was manually confirmed to be localized to the apical/basolateral membrane in kidney tissues. Furthermore, we collected 421 plasma membrane proteins from the RBCC database [36] and other sources [31], including our previous experimental pipelines [37][38][39]. We collected 521 proteins localizing to other membranes (Mitochondrial membrane, Endoplasmic reticulum, Lysosomal membrane etc) from UniProt [40].…”

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method

“…According to their importance, the detailed characterization of cell surface accessible segments is crucial for targeted therapy, such as immunotherapy [6] or the development of vaccines against particular infectious diseases [7]. Nevertheless, due to their relatively low abundance, compared with the other cellular proteins [8,9] and their specific physical-chemical properties (especially in the case of transmembrane proteins [10]), achieving their comprehensive identification and their extracellular accessible segments remains a difficult task.…”

“…To overcome this limitation, many high-throughput methods (mainly with mass spectrometry detection) have been developed for the characterization of CSPs and/or their specific regions [9,12]. These methods can be classified based on enrichment strategies preceding the MS analysis, which include ultracentrifugation to obtain membrane protein fractions [13], enzymatic treatment of cell surface to promote the extracellular peptide release [14], or partial proteolysis of surface proteins to cause new peptide termini that can be labeled and separated based on the modification [10]. Additional strategies are the attachment of cationic colloidal silica beads to the membranes to promote their isolation with increasing density [15] and the use of biotin-containing reagents with various chemical specificities [16][17][18][19].…”

“…The vast majority of the chemical modification strategies for CSPs utilize two different pipelines. Some methods target the extracellularly exposed post-translational modifications on the CSPs (as N-glycan moieties [20,21]), while other approaches modify the accessible/free side chains of various residues (lysine [10,19] or aspartic and glutamic acids [22]) on the outside region of the plasma membrane. N-glycopeptide enrichment offers a highly specific and selective method for the identification of extracellular segments of CSPs [23]; however, the frequency of the N-X-S/T/C motifs is lower than other reactive side chains containing residues [19].…”

“…The optimized method allows the characterization of the CSPs and the identification of several hundreds of extracellular regions belonging to various transmembrane proteins (TMPs, the most important subclass of the CSPs that have at least one transmembrane segment). Although their high-resolution structural determinations are constantly evolving by emerging new biotechnological and artificial intelligence tools such as Cryo-EM and natural language models utilized by AlphaFold2 [38], other bioinformatical [39] and experimental methods [10,40] still have a great role in topology characterization of TMPs (topology defines the number of transmembrane segments and the orientation of the connecting loop relative to the membrane). While most of the existing experimental approaches often produce few topology data for only one TMP, the work presented here can produce several data for dozens of proteins at the same time.…”