No abstract

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein–protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. Virotrap based interactome analysis of two N-terminal proteoforms originating from non-coding regions finally showed the functional potential of these novel proteins.

Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain protein concentrations that vastly exceed those of the EV proteome and contain particles in the same size and density range (e.g. protein aggregates and apolipoprotein particles). Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion and precipitation methods. Here, we describe an approach based on 300 kDa MWCO filtration, which allows processing of multiple samples in parallel within a reasonable timeframe and at moderate cost. We demonstrate that our strategy is capable of quantitatively retaining EV particles on filters, whilst allowing extensive washing with relatively high percentages of the mild detergent TWEEN-20. In addition, we provide evidence that the retained EVs can be recuperated from the filter for qualitative studies or can be directly lysed on the filter for the recovery of the EV protein cargo for proteome analysis. Applying this strategy on MCF7 conditioned medium using different percentages of serum, we observed dramatic changes in the EV proteome.

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