科学論文紹介

BioID-tagは免疫電顕に迫る解像度の顕微鏡として使える?

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論文タイトル: An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations (pubmed; Nature Commnucations)

ラボで紹介した論文が面白かったので、そのまま転載します。

Introduction

Proteomic and interactomic insights is essentaial to understand biological phenomena. Recently various techniques have been developed, and proximity labeling (such as BioID and APEX) is one of the hot topic.

In this paper the authors combined affinity purification coupled to mass spectrometry (AP-MS) and proximity labelling to define interaction distances. Further they developed MS microscopy the resolution of which is comparable to immunoEM.

Results

Calculation of interaction distances using AP-MS and BioID

First, they examined number of interactions. As expected, the total number of identified interactors as well as interactions per bait (or target protein) of BioID was higher than that of AP-MS (Fig. 3a, b). On average BioID and AP-MS captured 100 and 25 interactors, respectively. Of course, some interactors were overlapped between these two methods due to their complementary nature. In the case of PSA1 (Fig. 3c), 17 and 2 interactors were unique to BioID and AP-MS, respectively, and 17 interactors were overlapped.

Next, they defined interaction distances by using the normalized peptide spectrum matches (PSMs) to calculate the relative Euclidean distance between the baits and the preys using the formula below.

SQRT{(PSM[prey/BioID] – PSM[bait/BioID])^2 + (PSM[prey/AP-MS] – PSM[bait/AP-MS])^2}

As the example, they calculated relative interaction distances between CDK7 and TFIIH core components (Fig. 7a, f). Using the relative interaction distances, they tried to dock crystal structures of each component into the cryoEM reconstruction, and succeeded nicely like a puzzle (Fig. 7d).

MS microscopy using BioID

Further, the authors also developed MS microscopy. In this method, they used 14 proteins from different cellular compartments as references, and calculated localization scores. I skip the formula, because it is difficult to write down. But the formula is simple as well. As the example, they calculated localization scores of 6 proteins, and the result was well matched with immuno-staining (Fig.4).

Theoretically, the resolution of MS microscopy reaches 10–50 nm, which is 5–10-fold higher resolution than that of standard confocal microscope. You can imagine the performance of MS microscopy by remembering that the diameter of nucleosome is 10 nm. This is enough to examine the protein localization in sub-organelle level.

To benchmark the MS microscopy on sub-organelle analyses, they selected three well-documented mitochondrial proteins as references (outer mitochondrial membrane: TOM20; inner mitochondrial membrane space: SCO1; mitochondrial matrix: PDK1). They calculated localization scores for 13 proteins and defined their mitochondrial sub-organelle localization (Fig. 5). Using confocal microscopy, they could not define their sub-organelle localization (Fig. 5).

Discssion

It is not too much to say that the resolution of MS microscopy is comparable to that of immunoEM.
I again realized that BioID is a wonderful technique!