Jordan B. Burton, Ph.D.

Post-doctoral Research Fellow, Buck Institute for Research on Aging

Pattern Recognition plus Elute to Wash Ratios Identify Proteins of Interest in Proximity Labeling Experiments

Authors

Jordan B. Burton (Presenter)

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Nicholas J. Carruthers

Zhanjun Hou

Larry H. Matherly

Paul M. Stemmer

Introduction

Interpretating affinity selection experiments is complicated by high background leading to false positives. Particularly with streptavidin-based selection of biotinylated targets, even highly stringent washes with detergents leave high background proteomes. Using localization of organelle proteins by isotope tagging (LOPIT) maps helps in the interpretation of streptavidin affinity selection data through pattern recognition. A proximity labeling experiment with the Bir-A biotin ligase fused to the folate transporter was completed. Proteins of interest as potential interacting partners to the folate transporter were isolated using streptavidin beads with the final wash collected and evaluated along with the elute fractions. Mapping the subcellular localization of identified proteins and establishing a cut off for the elute to wash abundance ratio (E:W) simplifies biological interpretation.

Methods

HeLa cells stably transfected with plasmids for the PCFT-BirA fusion or for BirA alone were cultured in biotin free DMEM for 48 hours then with or without 50 uM biotin for 16 hr prior to harvest. Standard conditions were employed for cell lysis, lysate clarification and affinity selection on streptavidin beads. Elution differed from wash only in addition of 25 uM biotin and incubation at 65 C. Collected proteins were reduced with DTT, alkylated with IAA and digested with trypsin prior to LC-MS/MS on an orbitrap Fusion. For the proton-coupled folate transporter group (PCFT), the bait protein was expressed as a fusion with BirA. For the ground reference group (GR), BirA was expressed as an uncomplexed protein.

Results

The distribution pattern for proteins in the Elute fractions shows high density in the plasma membrane and cytosolic regions of the LOPIT plot as expected for the PCFT. Proteins in the LOPIT plot for the GR group are broadly distributed. 2403 of the 3249 proteins identified in the Wash and 887 of the 1220 proteins identified in the Elute were available for localization on LOPIT maps. The fold change for the Elute vs. Wash fractions was calculated for all analytes with at least 3 values for the 10 samples. Carboxylases are present in lower abundance in the Wash fractions than in the Elute fractions but they are detected in all Wash and Elute samples. Pyruvate carboxylase (P11498), propionyl-CoA carboxylase alpha chain (P05165), methylcrotonoyl-CoA carboxylase subunit alpha (Q96RQ3), and acetyl-CoA carboxylase 1 (Q13085) are used as endogenous positive controls for biotin-dependent selection. The E:W ratio of total carboxylase abundance establishes a minimum ratio necessary for any protein in the Elute fractions to be considered to have been selected due to biotinylation. For the Carboxylases this ratio ranged from 2.1 to 68.8 for the PCFT group and 6.7 to 83.9 for the GR group. Based on the carboxylase E:W ratios a cut off of 3X was selected to filter the data and decrease the occurrence of false positives. Proteins with larger fold changes between the Elute and the Wash fractions are more likely to have true interactions with the bait protein. The E:W cut off decreased the proteins of interest from 1220 to 238. The remaining proteins of interest were highly localized in the PM and Cytosol areas of the LOPIT plot for the PCFT group but were broadly distributed for the GR group.

Novel Aspect

Interpretation of proximity labeling data is facilitated by organellar profiling and Elute to Wash ratios using carboxylases as positive controls.

Availability

Poster

To view the poster, click here.

Paper

This poster was based off of our publication “Pattern Analysis of Organellar Maps for Interpretation of Proteomic Data”.

Paper Abstract Localization of organelle proteins by isotope tagging (LOPIT) maps are a coordinate-directed representation of proteome data that can aid in biological interpretation. Analysis of organellar association for proteins as displayed using LOPIT is evaluated and interpreted for two types of proteomic data sets. First, test and control group protein abundances and fold change data obtained in a proximity labeling experiment are plotted on a LOPIT map to evaluate the likelihood of true protein interactions. Selection of true positives based on co-localization of proteins in the organellar space is shown to be consistent with carboxylase enrichment which serves as a positive control for biotinylation in streptavidin affinity selected proteome data sets. The mapping in organellar space facilitates discrimination between the test and control groups and aids in identification of proteins of interest. The same representation of proteins in organellar space is used in the analysis of extracellular vesicle proteomes for which protein abundance and fold change data are evaluated. Vesicular protein organellar localization patterns provide information about the subcellular origin of the proteins in the samples which are isolates from the extracellular milieu. The organellar localization patterns are indicative of the provenance of the vesicular proteome origin and allow discrimination between proteomes prepared using different enrichment methods. The patterns in LOPIT displays are easy to understand and compare which aids in the biological interpretation of proteome data.

Keywords Proteomics; Pattern Analysis; Bioinformatics; Proximity Labeling; Exosomes