SPDesign: protein sequence designer based on structural sequence profile using ultrafast shape recognition.

Protein sequence design can provide valuable insights into biopharmaceuticals and disease treatments. Currently, most protein sequence design methods based on deep learning focus on network architecture optimization, while ignoring protein-specific physicochemical features. Inspired by the successful application of structure templates and pre-trained models in the protein structure prediction, we explored whether the representation of structural sequence profile can be used for protein sequence design. In this work, we propose SPDesign, a method for protein sequence design based on structural sequence profile using ultrafast shape recognition. Given an input backbone structure, SPDesign utilizes ultrafast shape recognition vectors to accelerate the search for similar protein structures in our in-house PAcluster80 structure database and then extracts the sequence profile through structure alignment. Combined with structural pre-trained knowledge and geometric features, they are further fed into an enhanced graph neural network for sequence prediction. The results show that SPDesign significantly outperforms the state-of-the-art methods, such as ProteinMPNN, Pifold and LM-Design, leading to 21.89%, 15.54% and 11.4% accuracy gains in sequence recovery rate on CATH 4.2 benchmark, respectively. Encouraging results also have been achieved on orphan and de novo (designed) benchmarks with few homologous sequences. Furthermore, analysis conducted by the PDBench tool suggests that SPDesign performs well in subdivided structures. More interestingly, we found that SPDesign can well reconstruct the sequences of some proteins that have similar structures but different sequences. Finally, the structural modeling verification experiment indicates that the sequences designed by SPDesign can fold into the native structures more accurately.

Mass Spectrometric De Novo Sequencing of Natural Peptides.

Natural peptides secreted under stress conditions by many organisms are bioactive molecules with a broad spectrum of activities. These molecules could become potential models for novel pharmaceuticals, to which bacteria, according to modern scientific concepts, do not have and cannot develop resistance. Taking this into consideration, it is necessary to clarify the amino acid sequences of such peptides. Here we describe our approach to de novo sequencing of amphibians' skin secretion peptides.

Introducing π-HelixNovo for practical large-scale de novo peptide sequencing.

De novo peptide sequencing is a promising approach for novel peptide discovery, highlighting the performance improvements for the state-of-the-art models. The quality of mass spectra often varies due to unexpected missing of certain ions, presenting a significant challenge in de novo peptide sequencing. Here, we use a novel concept of complementary spectra to enhance ion information of the experimental spectrum and demonstrate it through conceptual and practical analyses. Afterward, we design suitable encoders to encode the experimental spectrum and the corresponding complementary spectrum and propose a de novo sequencing model $\pi$-HelixNovo based on the Transformer architecture. We first demonstrated that $\pi$-HelixNovo outperforms other state-of-the-art models using a series of comparative experiments. Then, we utilized $\pi$-HelixNovo to de novo gut metaproteome peptides for the first time. The results show $\pi$-HelixNovo increases the identification coverage and accuracy of gut metaproteome and enhances the taxonomic resolution of gut metaproteome. We finally trained a powerful $\pi$-HelixNovo utilizing a larger training dataset, and as expected, $\pi$-HelixNovo achieves unprecedented performance, even for peptide-spectrum matches with never-before-seen peptide sequences. We also use the powerful $\pi$-HelixNovo to identify antibody peptides and multi-enzyme cleavage peptides, and $\pi$-HelixNovo is highly robust in these applications. Our results demonstrate the effectivity of the complementary spectrum and take a significant step forward in de novo peptide sequencing.

Bidirectional de novo peptide sequencing using a transformer model.

In proteomics, a crucial aspect is to identify peptide sequences. De novo sequencing methods have been widely employed to identify peptide sequences, and numerous tools have been proposed over the past two decades. Recently, deep learning approaches have been introduced for de novo sequencing. Previous methods focused on encoding tandem mass spectra and predicting peptide sequences from the first amino acid onwards. However, when predicting peptides using tandem mass spectra, the peptide sequence can be predicted not only from the first amino acid but also from the last amino acid due to the coexistence of b-ion (or a- or c-ion) and y-ion (or x- or z-ion) fragments in the tandem mass spectra. Therefore, it is essential to predict peptide sequences bidirectionally. Our approach, called NovoB, utilizes a Transformer model to predict peptide sequences bidirectionally, starting with both the first and last amino acids. In comparison to Casanovo, our method achieved an improvement of the average peptide-level accuracy rate of approximately 9.8% across all species.

PseAAC2Vec protein encoding for TCR protein sequence classification.

The classification and prediction of T-cell receptors (TCRs) protein sequences are of significant interest in understanding the immune system and developing personalized immunotherapies. In this study, we propose a novel approach using Pseudo Amino Acid Composition (PseAAC) protein encoding for accurate TCR protein sequence classification. The PseAAC2Vec encoding method captures the physicochemical properties of amino acids and their local sequence information, enabling the representation of protein sequences as fixed-length feature vectors. By incorporating physicochemical properties such as hydrophobicity, polarity, charge, molecular weight, and solvent accessibility, PseAAC2Vec provides a comprehensive and informative characterization of TCR protein sequences. To evaluate the effectiveness of the proposed PseAAC2Vec encoding approach, we assembled a large dataset of TCR protein sequences with annotated classes. We applied the PseAAC2Vec encoding scheme to each sequence and generated feature vectors based on a specified window size. Subsequently, we employed state-of-the-art machine learning algorithms, such as support vector machines (SVM) and random forests (RF), to classify the TCR protein sequences. Experimental results on the benchmark dataset demonstrated the superior performance of the PseAAC2Vec-based approach compared to existing methods. The PseAAC2Vec encoding effectively captures the discriminative patterns in TCR protein sequences, leading to improved classification accuracy and robustness. Furthermore, the encoding scheme showed promising results across different window sizes, indicating its adaptability to varying sequence contexts.

Deep learning-driven fragment ion series classification enables highly precise and sensitive de novo peptide sequencing.

Unlike for DNA and RNA, accurate and high-throughput sequencing methods for proteins are lacking, hindering the utility of proteomics in applications where the sequences are unknown including variant calling, neoepitope identification, and metaproteomics. We introduce Spectralis, a de novo peptide sequencing method for tandem mass spectrometry. Spectralis leverages several innovations including a convolutional neural network layer connecting peaks in spectra spaced by amino acid masses, proposing fragment ion series classification as a pivotal task for de novo peptide sequencing, and a peptide-spectrum confidence score. On spectra for which database search provided a ground truth, Spectralis surpassed 40% sensitivity at 90% precision, nearly doubling state-of-the-art sensitivity. Application to unidentified spectra confirmed its superiority and showcased its applicability to variant calling. Altogether, these algorithmic innovations and the substantial sensitivity increase in the high-precision range constitute an important step toward broadly applicable peptide sequencing.

Improving Signal and Transit Peptide Predictions Using AlphaFold2-predicted Protein Structures.

Many proteins contain cleavable signal or transit peptides that direct them to their final subcellular locations. Such peptides are usually predicted from sequence alone using methods such as TargetP 2.0 and SignalP 6.0. While these methods are usually very accurate, we show here that an analysis of a protein's AlphaFold2-predicted structure can often be used to identify false positive predictions. We start by showing that when given a protein's full-length sequence, AlphaFold2 builds experimentally annotated signal and transit peptides in orientations that point away from the main body of the protein. This indicates that AlphaFold2 correctly identifies that a signal is not destined to be part of the mature protein's structure and suggests, as a corollary, that predicted signals that AlphaFold2 folds with high confidence into the main body of the protein are likely to be false positives. To explore this idea, we analyzed predicted signal peptides in 48 proteomes made available in DeepMind's AlphaFold2 database (https://alphafold.ebi.ac.uk). Applying TargetP 2.0 and SignalP 6.0 to the 561,562 proteins in the database results in 95,236 being predicted to contain a cleavable signal or transit peptide. In 95.1% of these cases, the AlphaFold2 structure of the full-length protein is fully consistent with the prediction of TargetP 2.0 or SignalP 6.0. In the remaining 4.9% of cases where the AlphaFold2 structure does not appear consistent with the prediction, the signal is often only predicted with low confidence. The potential false positives identified here may be useful for training even more accurate signal prediction methods.

FunPredCATH: An ensemble method for predicting protein function using CATH.

MOTIVATION: The growth of unannotated proteins in UniProt increases at a very high rate every year due to more efficient sequencing methods. However, the experimental annotation of proteins is a lengthy and expensive process. Using computational techniques to narrow the search can speed up the process by providing highly specific Gene Ontology (GO) terms. METHODOLOGY: We propose an ensemble approach that combines three generic base predictors that predict Gene Ontology (BP, CC and MF) terms from sequences across different species. We train our models on UniProtGOA annotation data and use the CATH domain resources to identify the protein families. We then calculate a score based on the prevalence of individual GO terms in the functional families that is then used as an indicator of confidence when assigning the GO term to an uncharacterised protein. METHODS: In the ensemble, we use a statistics-based method that scores the occurrence of GO terms in a CATH FunFam against a background set of proteins annotated by the same GO term. We also developed a set-based method that uses Set Intersection and Set Union to score the occurrence of GO terms within the same CATH FunFam. Finally, we also use FunFams-Plus, a predictor method developed by the Orengo Group at UCL to predict GO terms for uncharacterised proteins in the CAFA3 challenge. EVALUATION: We evaluated the methods against the CAFA3 benchmark and DomFun. We used the Precision, Recall and Fmax metrics and the benchmark datasets that are used in CAFA3 to evaluate our models and compare them to the CAFA3 results. Our results show that FunPredCATH compares well with top CAFA methods in the different ontologies and benchmarks. CONTRIBUTIONS: FunPredCATH compares well with other prediction methods on CAFA3, and the ensemble approach outperforms the base methods. We show that non-IEA models obtain higher Fmax scores than the IEA counterparts, while the models including IEA annotations have higher coverage at the expense of a lower Fmax score.

Accurate de novo peptide sequencing using fully convolutional neural networks.

De novo peptide sequencing, which does not rely on a comprehensive target sequence database, provides us with a way to identify novel peptides from tandem mass spectra. However, current de novo sequencing algorithms suffer from low accuracy and coverage, which hinders their application in proteomics. In this paper, we present PepNet, a fully convolutional neural network for high accuracy de novo peptide sequencing. PepNet takes an MS/MS spectrum (represented as a high-dimensional vector) as input, and outputs the optimal peptide sequence along with its confidence score. The PepNet model is trained using a total of 3 million high-energy collisional dissociation MS/MS spectra from multiple human peptide spectral libraries. Evaluation results show that PepNet significantly outperforms current best-performing de novo sequencing algorithms (e.g. PointNovo and DeepNovo) in both peptide-level accuracy and positional-level accuracy. PepNet can sequence a large fraction of spectra that were not identified by database search engines, and thus could be used as a complementary tool to database search engines for peptide identification in proteomics. In addition, PepNet runs around 3x and 7x faster than PointNovo and DeepNovo on GPUs, respectively, thus being more suitable for the analysis of large-scale proteomics data.

Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

PreHom-PCLM: protein remote homology detection by combing motifs and protein cubic language model.

Protein remote homology detection is essential for structure prediction, function prediction, disease mechanism understanding, etc. The remote homology relationship depends on multiple protein properties, such as structural information and local sequence patterns. Previous studies have shown the challenges for predicting remote homology relationship by protein features at sequence level (e.g. position-specific score matrix). Protein motifs have been used in structure and function analysis due to their unique sequence patterns and implied structural information. Therefore, designing a usable architecture to fuse multiple protein properties based on motifs is urgently needed to improve protein remote homology detection performance. To make full use of the characteristics of motifs, we employed the language model called the protein cubic language model (PCLM). It combines multiple properties by constructing a motif-based neural network. Based on the PCLM, we proposed a predictor called PreHom-PCLM by extracting and fusing multiple motif features for protein remote homology detection. PreHom-PCLM outperforms the other state-of-the-art methods on the test set and independent test set. Experimental results further prove the effectiveness of multiple features fused by PreHom-PCLM for remote homology detection. Furthermore, the protein features derived from the PreHom-PCLM show strong discriminative power for proteins from different structural classes in the high-dimensional space. Availability and Implementation: http://bliulab.net/PreHom-PCLM.

Novel Proteoform Discovery by Precise Semi-De Novo Sequencing of Novel Junction Peptides.

Alternative splicing allows a small number of human genes to encode large amounts of proteoforms that play essential roles in normal and disease physiology. Some low-abundance proteoforms may remain undiscovered due to limited detection and analysis capabilities. Peptides coencoded by novel exons and annotated exons separated by introns are called novel junction peptides, which are the key to identifying novel proteoforms. Traditional de novo sequencing does not take into account the specificity in the composition of the novel junction peptide and is therefore not as accurate. We first developed a novel de novo sequencing algorithm, CNovo, which outperformed the mainstream PEAKS and Novor in all six test sets. We then built on CNovo to develop a semi-de novo sequencing algorithm, SpliceNovo, specifically for identifying novel junction peptides. SpliceNovo identifies junction peptides with much higher accuracy than CNovo, CJunction, PEAKS, and Novor. Of course, it is also possible to replace the built-in CNovo in SpliceNovo with other more accurate de novo sequencing algorithms to further improve its performance. We also successfully identified and validated two novel proteoforms of the human EIF4G1 and ELAVL1 genes by SpliceNovo. Our results significantly improve the ability to discover novel proteoforms through de novo sequencing.

Facile Method for High-throughput Identification of Stabilizing Mutations.

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.

Algorithms for de-novo sequencing of peptides by tandem mass spectrometry: A review.

Peptide sequencing is of great significance to fundamental and applied research in the fields such as chemical, biological, medicinal and pharmaceutical sciences. With the rapid development of mass spectrometry and sequencing algorithms, de-novo peptide sequencing using tandem mass spectrometry (MS/MS) has become the main method for determining amino acid sequences of novel and unknown peptides. Advanced algorithms allow the amino acid sequence information to be accurately obtained from MS/MS spectra in short time. In this review, algorithms from exhaustive search to the state-of-art machine learning and neural network for high-throughput and automated de-novo sequencing are introduced and compared. Impacts of datasets on algorithm performance are highlighted. The current limitations and promising direction of de-novo peptide sequencing are also discussed in this review.

BioSeq-Diabolo: Biological sequence similarity analysis using Diabolo.

As the key for biological sequence structure and function prediction, disease diagnosis and treatment, biological sequence similarity analysis has attracted more and more attentions. However, the exiting computational methods failed to accurately analyse the biological sequence similarities because of the various data types (DNA, RNA, protein, disease, etc) and their low sequence similarities (remote homology). Therefore, new concepts and techniques are desired to solve this challenging problem. Biological sequences (DNA, RNA and protein sequences) can be considered as the sentences of "the book of life", and their similarities can be considered as the biological language semantics (BLS). In this study, we are seeking the semantics analysis techniques derived from the natural language processing (NLP) to comprehensively and accurately analyse the biological sequence similarities. 27 semantics analysis methods derived from NLP were introduced to analyse biological sequence similarities, bringing new concepts and techniques to biological sequence similarity analysis. Experimental results show that these semantics analysis methods are able to facilitate the development of protein remote homology detection, circRNA-disease associations identification and protein function annotation, achieving better performance than the other state-of-the-art predictors in the related fields. Based on these semantics analysis methods, a platform called BioSeq-Diabolo has been constructed, which is named after a popular traditional sport in China. The users only need to input the embeddings of the biological sequence data. BioSeq-Diabolo will intelligently identify the task, and then accurately analyse the biological sequence similarities based on biological language semantics. BioSeq-Diabolo will integrate different biological sequence similarities in a supervised manner by using Learning to Rank (LTR), and the performance of the constructed methods will be evaluated and analysed so as to recommend the best methods for the users. The web server and stand-alone package of BioSeq-Diabolo can be accessed at http://bliulab.net/BioSeq-Diabolo/server/.

iQDeep: an integrated web server for protein scoring using multiscale deep learning models.

The remarkable recent advances in protein structure prediction have enabled computational modeling of protein structures with considerably higher accuracy than ever before. While state-of-the-art structure prediction methods provide self-assessment confidence scores of their own predictions, an independent and open-access system for protein scoring is still needed that can be applied to a broad range of predictive modeling scenarios. Here, we present iQDeep, an integrated and highly customizable web server for protein scoring, freely available at http://fusion.cs.vt.edu/iQDeep. The underlying method of iQDeep employs multiscale deep residual neural networks (ResNets) to perform residue-level error classifications, and then probabilistically combines the error classifications for protein scoring. By adjusting the error resolutions, our method can reliably estimate the standard- or high-accuracy variants of the Global Distance Test metric for versatile protein scoring. The performance of the method has been extensively tested and compared against the state-of-the-art approaches in multiple rounds of Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments including benchmark assessment in CASP12 and CASP13 as well as blind evaluation in CASP14. The iQDeep web server offers a number of convenient features, including (i) the choice of individual and batch processing modes; (ii) an interactive and privacy-preserving web interface for automated job submission, tracking, and results retrieval; (iii) web-based quantitative and visual analyses of the results including overall estimated score and its residue-wise breakdown along with agreements between various sequence- and structural-level features; (iv) extensive help information on job submission and results interpretation via web-based tutorial and help tooltips.

Protein length distribution is remarkably uniform across the tree of life.

BACKGROUND: In every living species, the function of a protein depends on its organization of structural domains, and the length of a protein is a direct reflection of this. Because every species evolved under different evolutionary pressures, the protein length distribution, much like other genomic features, is expected to vary across species but has so far been scarcely studied. RESULTS: Here we evaluate this diversity by comparing protein length distribution across 2326 species (1688 bacteria, 153 archaea, and 485 eukaryotes). We find that proteins tend to be on average slightly longer in eukaryotes than in bacteria or archaea, but that the variation of length distribution across species is low, especially compared to the variation of other genomic features (genome size, number of proteins, gene length, GC content, isoelectric points of proteins). Moreover, most cases of atypical protein length distribution appear to be due to artifactual gene annotation, suggesting the actual variation of protein length distribution across species is even smaller. CONCLUSIONS: These results open the way for developing a genome annotation quality metric based on protein length distribution to complement conventional quality measures. Overall, our findings show that protein length distribution between living species is more uniform than previously thought. Furthermore, we also provide evidence for a universal selection on protein length, yet its mechanism and fitness effect remain intriguing open questions.

Enzyme function prediction using contrastive learning.

Enzyme function annotation is a fundamental challenge, and numerous computational tools have been developed. However, most of these tools cannot accurately predict functional annotations, such as enzyme commission (EC) number, for less-studied proteins or those with previously uncharacterized functions or multiple activities. We present a machine learning algorithm named CLEAN (contrastive learning-enabled enzyme annotation) to assign EC numbers to enzymes with better accuracy, reliability, and sensitivity compared with the state-of-the-art tool BLASTp. The contrastive learning framework empowers CLEAN to confidently (i) annotate understudied enzymes, (ii) correct mislabeled enzymes, and (iii) identify promiscuous enzymes with two or more EC numbers-functions that we demonstrate by systematic in silico and in vitro experiments. We anticipate that this tool will be widely used for predicting the functions of uncharacterized enzymes, thereby advancing many fields, such as genomics, synthetic biology, and biocatalysis.

Homology Modeling in the Twilight Zone: Improved Accuracy by Sequence Space Analysis.

The analysis of the relationship between sequence and structure similarities during the evolution of a protein family has revealed a limit of sequence divergence for which structural conservation can be confidently assumed and homology modeling is reliable. Below this limit, the twilight zone corresponds to sequence divergence for which homology modeling becomes increasingly difficult and requires specific methods. Either with conventional threading methods or with recent deep learning methods, such as AlphaFold, the challenge relies on the identification of a template that shares not only a common ancestor (homology) but also a conserved structure with the query. As both homology and structural conservation are transitive properties, mining of sequence databases followed by multidimensional scaling (MDS) of the query sequence space can reveal intermediary sequences to infer homology and structural conservation between the query and the template. Here, as a case study, we studied the plethodontid receptivity factor isoform 1 (PRF1) from Plethodon jordani, a member of a pheromone protein family present only in lungless salamanders and weakly related to cytokines of the IL6 family. A variety of conventional threading methods led to the cytokine CNTF as a template. Sequence mining, followed by phylogenetic and MDS analysis, provided missing links between PRF1 and CNTF and allowed reliable homology modeling. In addition, we compared automated models obtained from web servers to a customized model to show how modeling can be improved by expert information.

Illuminating the "Twilight Zone": Advances in Difficult Protein Modeling.

Homology modeling was long considered a method of choice in tertiary protein structure prediction. However, it used to provide models of acceptable quality only when templates with appreciable sequence identity with a target could be found. The threshold value was long assumed to be around 20-30%. Below this level, obtained sequence identity was getting dangerously close to values that can be obtained by chance, after aligning any random, unrelated sequences. In these cases, other approaches, including ab initio folding simulations or fragment assembly, were usually employed. The most recent editions of the CASP and CAMEO community-wide modeling methods assessment have brought some surprising outcomes, proving that much more clues can be inferred from protein sequence analyses than previously thought. In this chapter, we focus on recent advances in the field of difficult protein modeling, pushing the threshold deep into the "twilight zone", with particular attention devoted to improvements in applications of machine learning and model evaluation.