Identification of Canonical and Alternative Proteins by Mass Spectrometry

Alternative proteins (AltProts) produced from short open reading frames have been missed when genomes were annotated primarily due to their small sizes. In addition, their limited size and low abundance further challenge their identification using conventional proteomics. Several specific workflows have been developed to identify AltProts by mass spectrometry. However, the different protocols rely on the separation of AltProts from canonical proteins, thus preventing concomitant identification of both types of molecules within a single sample. Here, we present an experimental strategy for simultaneous identification of both canonical proteins and AltProts within a single mass spectrometry analysis. This approach includes a simple protein extraction method and an analysis pipeline dedicated to the identification of AltProts.