DOI: 10.25345/c5vq2sm8q
title: MassIVE MSV000093145 - Co-translational assembly of NatA Complex
title: Project Description Protein-protein interactions are fundamental to all cellular activities, with the ribosome increasingly recognized as a central platform steering the dynamics of nascent-chain interactions. In our focus on conserved N-terminal acetyltransferases (NATs), we identified distinct co-translational assembly pathways. This distinction persisted even among highly homologous subunits, with some playing contrasting roles. Notably, we found that only a few residues act as hotspots, initiating co-translational assembly interactions as they emerge from the ribosome exit tunnel. The significance of these hotspots was further validated in vivo through N-terminomics studies. Sample Processing Protocol WT, NAA10 knockout, NAA15 knockout, mutant NAA15 (R354E, R355E) and mutant NAA15 (R354A, R355A) yeast cells were grown in appropriate SD media and plates to allow the selective growth of each strain. Single colonies of each strain were streaked onto 5mL YPD media and grown overnight at 30C, 250 rpm. Cultures were than diluted 25 fold in 50mL fresh YPD and grown until OD600=1. Cells were harvested by centrifugation at 4000 rpm for 5min in RT. Yeast pellets were washed 3 times with ice-cold water and flash-frozen in liquid nitrogen and stored in -80C. Frozen yeast pellets were dissolved in 250uL 1% sodium deoxycholate in 100mM HEPES pH-8.0, and incubated at 95C for 10min. Protein concentration was determined using the BCA method. The samples were then subjected to a denaturation, reduction, and alkylation processes. The NAA10/15 knockout and NAA15 mutant samples were labeled with light formaldehyde (C12H2) and WT sample labeled with heavy formaldehyde (C13D2). Finally, equal protein amounts of light-labelled NAA knockout/mutants and heavy-labelled WT samples were mixed the subjected to N-terminal enrichment based on HYTANE method using the protocol described in DOI: 10.1016/bs.mie.2023.04.003. Following enrichment and desalting the samples were analyzed by a tandem mass spectrometry using using Thermo Q-Exactive Plus Orbitrap coupled to Easy nano-LC 1000 capillary HPLC. Enriched Terminal peptides were resolved on a homemade reverse phase capillary 30cm-long and 75um-diamter, packed with 3.5um silica using ReproSil-Pur C18-AQ resin (Dr. Maisch GmbH). Elution was preformed with a 120 min linear gradient of Acetonitrile 5-28% (in 0.1% formic acid), followed by 15 minutes wash with 95% Acetonitrile (in 0.1% formic acid), with all flow rates set to 0.15uL/min. MS was conducted in a data-dependent acquisition mode for positive ions in an m/z range of 300-1500, with 70,000 resolution for MS1 and 17500 resolution for MS2. the 10 most dominant ions selected from the MS1 scan were fragmented using high energy collisional dissociation (HCD) with 25 normalized collision energy (NCE). Data Processing Protocol Data analysis was performed using the Trans Proteomic Pipline (version 6.1). Peptide search was done using MSFragger- version 3.7 via FragPipe version 19.1. The reset of the analysis was conducted for only N-terminal acetylated peptides as described in data analysis section of the protocol in DOI: 10.1016/bs.mie.2023.04.003. Peptide scoring was done using PeptideProphet at a false discovery rate of 1%. All data were searched against Uniprot Yeast proteome (UP000002311) containing 6090 sequences (downloaded April 2023) supplemented with known contaminant sequences and decoy sequences (via FragPipe 19.1).