A Method to Estimate the Distribution of Proteins across Multiple Compartments Using Data from Quantitative Proteomics Subcellular Fractionation Experiments

Abstract

Knowledge of cellular location is key to understanding the biological function of proteins. One commonly used large-scale method to assign cellular locations is subcellular fractionation, followed by quantitative mass spectrometry to identify proteins and estimate their relative distribution among centrifugation fractions. In most of such subcellular proteomics studies, each protein is assigned to a single cellular location by comparing its distribution to those of a set of single-compartment reference proteins. However, in many cases, proteins reside in multiple compartments. To accurately determine the localization of such proteins, we previously introduced constrained proportional assignment (CPA), a method that assigns each protein a fractional residence over all reference compartments (Jadot et al. Mol. Cell Proteomics 2017, 16(2), 194−212. ). In this Article, we describe the principles underlying CPA, as well as data transformations to improve accuracy of assignment of proteins and protein isoforms, and a suite of R-based programs to implement CPA and related procedures for analysis of subcellular proteomics data. We include a demonstration data set that used isobariclabeling mass spectrometry to analyze rat liver fractions. In addition, we describe how these programs can be readily modified by users to accommodate a wide variety of experimental designs and methods for protein quantitation.

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Clean-up Techniques

“…This result is in line with previous studies, which have shown that proteins can be located and distributed within multiple cellular compartments, e.g., the cytoplasmic fraction can contain cytoplasmic vesicles that may map to membranous-cellular components. 32,[52][53][54][55][56][57][58] Yet, these results, in combination with the Western blots presented in Figure S1, demonstrate that the subcellular fractionation was successful because for most of the subcellular fractions, increases were seen in the identified proteins associated with that cellular component.…”