title: The effect of organelle discovery upon sub-cellular protein localisation.
abstract: Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein-organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein-organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein-organelle membership from quantitative MS experiments.
  
  
  CONCLUSIONS
  Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in silico to known protein-organelle associations via supervised machine learning algorithms, to create classifiers that associate unannotated proteins to specific organelles. These strategies are prone to error, however, if organelle-related groupings present in experimental output are not represented, for example owing to the lack of existing annotation, when creating the protein-organelle mapping. Here, the application of a phenotype discovery approach to LOPIT gradient-based MS data identifies candidate organelle phenotypes for further evaluation in an unbiased fashion. Software implementation and usage guidelines are provided for application to wider protein-organelle association experiments. In the wider context, semi-supervised organelle discovery is discussed as a paradigm with which to generate new protein annotations from MS-based organelle proteomics experiments.
authors.affiliation: Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, CB2 1QR, UK.
authors.forename: L M
authors.initials: LM
authors.lastname: Breckels
authors.forename: L
authors.initials: L
authors.lastname: Gatto
authors.forename: A
authors.initials: A
authors.lastname: Christoforou
authors.forename: A J
authors.initials: AJ
authors.lastname: Groen
authors.forename: K S
authors.initials: KS
authors.lastname: Lilley
authors.forename: M W B
authors.initials: MW
authors.lastname: Trotter
mesh_terms: D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015388:Organelles / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D029721:Drosophila Proteins
keywords: Assignment; Machine-learning; Organelle; Prediction; Protein; Semi-supervised