Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks

Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP–MS and BioID data has been shown to comprehensively characterize a protein’s molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP–MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform (http://proteomics.fi) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP–MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.



Proteomics Review

Thyroid Review

Thyroid nodules: diagnostic evaluation based on thyroid cancer risk assessment

Thyroid nodules are extremely common and can be detected by sensitive imaging in more than 60% of the general population. They are often identified in patients without symptoms who are undergoing evaluation for other medical complaints. Indiscriminate evaluation of thyroid nodules with thyroid biopsy could cause a harmful epidemic of diagnoses of thyroid cancer, but inadequate selection of thyroid nodules for biopsy can lead to missed diagnoses of clinically relevant thyroid cancer. Recent clinical guidelines advocate a more conservative approach in the evaluation of thyroid nodules based on risk assessment for thyroid cancer, as determined by clinical and ultrasound features to guide the need for biopsy. Moreover, newer evidence suggests that for patients with indeterminate thyroid biopsy results, a combined assessment including the initial ultrasound risk stratification or other ancillary testing (molecular markers, second opinion on thyroid cytology) can further clarify the risk of thyroid cancer and the management strategies. This review summarizes the clinical importance of adequate evaluation of thyroid nodules, focuses on the clinical evidence for diagnostic tests that can clarify the risk of thyroid cancer, and highlights the importance of considering the patient’s values and preferences when deciding on management strategies in the setting of uncertainty about the risk of thyroid cancer.

Thyroid surgery for differentiated thyroid cancer — recent advances and future directions

Population-based studies have demonstrated that an increasing number of incidental thyroid nodules are being identified. The corresponding increase in thyroid-based diagnostic procedures, such as fine-needle aspiration biopsy, has in part led to an increase in the diagnoses of thyroid cancers and to more thyroid surgeries being performed. Small papillary thyroid cancers account for most of this increase in diagnoses. These cancers are considered to be low risk because of the excellent patient outcomes, with a 5-year disease-specific survival of >98%. As a result, controversy remains regarding the optimal management of newly diagnosed differentiated thyroid cancer, as the complications related to thyroidectomy (primarily recurrent laryngeal nerve injury and hypoparathyroidism) have considerable effects on patient quality of life. This Review highlights current debates, including undertaking active surveillance versus thyroid surgery for papillary thyroid microcarcinoma, the extent of thyroid surgery and lymphadenectomy for low-risk differentiated thyroid cancer, and the use of molecular testing to guide decision-making about whether surgery is required and the extent of the initial operation. This Review includes a discussion of current consensus guideline recommendations regarding these topics in patients with differentiated thyroid cancer. Additionally, innovative thyroidectomy techniques (including robotic and transoral approaches) are discussed, with an emphasis on patient preferences around decision-making and outcomes following thyroidectomy.


Software & Database