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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

Knowledge of intracellular location can provide important insights into the function of proteins and their respective organelles, and there is interest in combining classical subcellular fractionation with quantitative mass spectrometry to create global cellular maps. To evaluate mass spectrometric approaches specifically for this application, we analyzed rat liver differential centrifugation and Nycodenz density gradient subcellular fractions by tandem mass tag (TMT) isobaric labeling with reporter ion measurement at the MS2 and MS3 level and with two different label-free peak integration approaches, MS1 and data independent acquisition (DIA). TMT-MS2 provided the greatest proteome coverage, but ratio compression from contaminating background ions resulted in a narrower accurate dynamic range compared to TMT-MS3, MS1, and DIA, which were similar. Using a protein clustering approach to evaluate data quality by assignment of reference proteins to their correct compartments, all methods performed well, with isobaric labeling approaches providing the highest quality localization. Finally, TMT-MS2 gave the lowest percentage of missing quantifiable data when analyzing orthogonal fractionation methods containing overlapping proteomes. In summary, despite inaccuracies resulting from ratio compression, data obtained by TMT-MS2 assigned protein localization as well as other methods but achieved the highest proteome coverage with the lowest proportion of missing values.

Clinical trials have been conducted for the neuronal ceroid lipofuscinoses (NCLs), a group of neurodegenerative lysosomal diseases that primarily affect children. Whereas clinical rating systems will evaluate long-term efficacy, biomarkers to measure short-term response to treatment would be extremely valuable. To identify candidate biomarkers, we analyzed autopsy brain and matching CSF samples from controls and three genetically distinct NCLs due to deficiencies in palmitoyl protein thioesterase 1 (CLN1 disease), tripeptidyl peptidase 1 (CLN2 disease), and CLN3 protein (CLN3 disease). Proteomic and biochemical methods were used to analyze lysosomal proteins, and, in general, we find that changes in protein expression compared with control were most similar between CLN2 disease and CLN3 disease. This is consistent with previous observations of biochemical similarities between these diseases. We also conducted unbiased proteomic analyses of CSF and brain using isobaric labeling/quantitative mass spectrometry. Significant alterations in protein expression were identified in each NCL, including reduced STXBP1 in CLN1 disease brain. Given the confounding variable of post-mortem changes, additional validation is required, but this study provides a useful starting set of candidate NCL biomarkers for further evaluation.