A short open reading frame (sORFs) constitutes ≤ 300 bases, encoding a microprotein or sORF-encoded protein (SEP) which comprises ≤ 100 amino acids. Traditionally dismissed by genome annotation pipelines as meaningless noise, sORFs were found to possess coding potential with ribosome profiling (RIBO-Seq), which unveiled sORF-based transcripts at various genome locations. Nonetheless, the existence of corresponding microproteins that are stable and functional was little substantiated by experimental evidence initially. With recent advancements in multi-omics, the identification, validation, and functional characterisation of sORFs and microproteins have become feasible. In this review, we discuss the history and development of an emerging research field of sORFs and microproteins. In particular, we focus on an array of bioinformatics and OMICS approaches used for predicting, sequencing, validating, and characterizing these recently discovered entities. These strategies include RIBO-Seq which detects sORF transcripts via ribosome footprints, and mass spectrometry (MS)-based proteomics for sequencing the resultant microproteins. Subsequently, our discussion extends to the functional characterisation of microproteins by incorporating CRISPR/Cas9 screen and protein–protein interaction (PPI) studies. Our review discusses not only detection methodologies, but we also highlight on the challenges and potential solutions in identifying and validating sORFs and their microproteins. The novelty of this review lies within its validation for the functional role of microproteins, which could contribute towards the future landscape of microproteomics.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an emergent technology that has been increasingly adopted in cancer research. MALDI imaging is capable of providing global molecular mapping of the abundance and spatial information of biomolecules directly in the tissues without labeling. It enables the characterization of a wide spectrum of analytes, including proteins, peptides, glycans, lipids, drugs, and metabolites and is well suited for both discovery and targeted analysis. An advantage of MALDI imaging is that it maintains tissue integrity, which allows correlation with histological features. It has proven to be a valuable tool for probing tumor heterogeneity and has been increasingly applied to interrogate molecular events associated with cancer. It provides unique insights into both the molecular content and spatial details that are not accessible by other techniques, and it has allowed considerable progress in the field of cancer research. In this review, we first provide an overview of the MALDI imaging workflow and approach. We then highlight some useful applications in various niches of cancer research, followed by a discussion of the challenges, recent developments and future prospect of this technique in the field.