Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics

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Highlights

• •Analysis of product ions produced by 213 nm UVPD is used to refine database search.
• •A product ion at the N-terminus of Pro, y-2, is observed in 213 nm UVPD spectra.
• •213 nm UVPD provides more complete proteoform characterization than HCD.
• •HCD and 213 nm UVPD are complementary fragmentation methods for proteoforms <30 kDa.

     

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.     

The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.     

A peptidoform based proteomic strategy for studying functions of post-translational modifications

Protein post-translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the produced proteoforms. In this Viewpoint, we firstly reviewed the concepts of proteoform and peptidoform. We show that many of the current PTM biological investigation and annotation studies largely follow a PTM site-specific rather than proteoform-specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy has the potential to provide more directly relevant information to learn the PTM site-specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in future bottom-up proteomic studies.     

FusionPro,a Versatile Proteogenomic Tool for Identification of Novel Fusion Transcripts and Their Potential Translation Products in Cancer Cells,

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Highlights

• •FusionPro, a versatile tool for studying fusion proteoforms, has been developed.
• •Fusion peptides were identified against a customized database built by FusionPro.
• •Types and features of fusion proteoforms were efficiently predicted by FusionPro.

     

Positional proteomics reveals differences in N‐terminal proteoform stability

To understand the impact of alternative translation initiation on a proteome, we performed a proteome‐wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N‐terminal proteoforms of individual genes. By combining pulsed SILAC with N‐terminal COFRADIC, we monitored the stability of 1,941 human N‐terminal proteoforms, including 147 N‐terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. N‐terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N‐terminal amino acid identity or truncation length. We discovered that the removal of initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms, while susceptibility for N‐terminal acetylation did not seem to influence protein turnover rates. Taken together, our findings reveal differences in protein stability between N‐terminal proteoforms and point to a role for alternative translation initiation and co‐translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis.     

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.