New insight into interactome of plant NDPKs
Interactions between metabolites and proteins play an integral role in all cellular functions. Here we describe an affinity-purification (AP) approach in combination with LC/MS-based metabolomics and proteomics that allows, to our knowledge for the first time, to analyse protein‒metabolite and protein‒protein interactions simultaneously in plant systems. More specifically, we examined protein and small-molecule partners of the three (of five) nucleoside diphosphate kinases present in the Arabidopsis genome (NDPK1‒3). The bona fide role of NDPKs is the exchange of terminal phosphate groups between nucleoside di- (NDP) and triphosphates (NTP). However, other functions have been reported, which likely depend on both the proteins and small molecules specifically interacting with the NDPK. Using our approach we identified 23, 17, and 8 novel protein partners of NDPK1, NDPK2, and NDPK3, respectively, with nucleotide-dependent proteins such as actin and adenosine kinase 2 being enriched. Particularly interesting, however, was the co-elution of glutathione S-transferases (GSTs) and reduced glutathione (GSH) with the affinity-purified NDPK1 complexes. Following up on this finding, we could demonstrate that NDPK1 undergoes glutathionylation, opening a new paradigm of NDPK regulation in plants. The described results extend our knowledge of NDPKs, the key enzymes regulating NDP/NTP homeostasis.
Sample Processing Protocol
Proteomic analysis was based on previous work of Olsen et al. (2004) and technical manual TM390 Trypsin/Lys-C Mix, Promega. In principle, protein fractions were subjected to enzymatic digestion prior to LC/MS/MS analysis. Protein pellets derived from metabolite extraction were dissolved in 40 mM ammonium bicarbonate (40 mM AmBic buffer) containing 6 M urea / 2 M thiourea, pH 8 (Olsen et al., 2004). Protein concentration was determined with the Bradford assay (Bradford, 1976). 100 µg of protein in 46 µl denaturation buffer (AmBic buffer pH 8.0, 2 M thiourea, 6 M urea) was treated with 2 µl of reduction buffer (50 mM DTT) and incubated for 30 min at RT. Then, 2 µl of alkylation buffer (150 mM iodoacetamide) was added to the sample and the mixture was incubated for 20 min at RT in the dark. Next, 30 µl of 40 mM AmBic buffer and 20 µl of LysC/Trypsin Mix were added and the sample was incubated for 4 h at 37 °C. After that, samples were diluted with 300 µl of 40 mM AmBic buffer and incubation continued at 37 °C overnight. Samples were acidified with 2 % trifluoroacetic acid (TFA) to pH < 2 and proteins were desalted using Finisterre C18 SPE columns (Teknokroma™) as follows. The column was washed with 1 ml of 100 % MeOH, 1 ml of 80 % acetonitrile, 0.1 % TFA (water solution), and equilibrated with 2 × 1 ml of 0.1 % TFA (water solution). Samples were loaded on the columns; tubes were further washed with 200 µl of 0.1 % TFA and loaded on the columns. Columns were washed with 2 × 1 ml of 0.1 % TFA and peptides were slowly eluted with 800 µl of 60 % acetonitrile, 0.1 % TFA. Peptides were dried using a centrifugal evaporator and stored at ‒80 °C. To analyse peptide samples we used an LC/MS system consisting of nano liquid chromatography (Proxeon EASY-nLC 1000, Thermo Fisher Scientific) with a reversed-phase column (C18, Acclaim PepMap RLSC, 75 µm, 15 cm, Thermo Fisher Scientific) connected to a Q Exactive Orbitrap Plus MS (Thermo Fisher Scientific). Dried peptides were re-suspended in 50 µl buffer A (3 % v/v acetonitrile (ACN), 0.1 % v/v formic acid). 3-µl samples were separated by reverse-phase nano liquid chromatography using buffer A and buffer B (63 % v/v ACN, 0.1 % v/v formic acid). The gradient ramped from 3 % ACN to 15 % ACN over 20 min, then to 30 % ACN in 10 min, followed by a 10-min washout with 60 % ACN. The flow rate was 300 nl min‒1 and the column was equilibrated with 5 µl of buffer A in between samples. The MS was run using a data-dependent MS/MS method with the following settings: full scans were acquired at a resolution of 70,000, AGC target of 3×106 ions, maximum injection time of 100 msec and a m/z ranging from 300 to 1,600. A maximum of 15 MS/MS scans were acquired per full scan (top 15) at a resolution of 17,500, AGC target of 105, maximum injection time of 100 ms, underfill ratio of 20 %, with an isolation window of 1.6 m/z and a m/z ranging from 200 to 2,000. Apex trigger was on (6 to 20 sec) and a dynamic exclusion set to 15 sec. Charge exclusion of charges 1 and >5 was on.
Data Processing Protocol
Data analysis was performed using MaxQuant software with integrated Andromeda peptide search engine (Cox and Mann, 2008; Cox et al., 2011) using default settings. The Uniprot database was downloaded on 15 March 2017 from http://www.uniprot.org/proteomes/UP000006548 as fasta (canonical & isoform) with all protein entries (33,037), last modified 18 December 2016. Search included also contaminant database (ftp://ftp.thegpm.org/fasta/cRAP). Peptides with at least seven amino acids were taken into account with both the peptide and protein FDR set to 1 % (see Table S5 for “parameters.txt” output file of MaxQuant analysis). Detailed information about all identified protein groups, including intensities, number of unique peptides, and score is included in Table S4 (see also Table S6 and Figure S2 for a general overview of data and replicate quality). Identified protein groups with less than two unique peptides and present at least in one technical replica of EV and/or blank controls were excluded from the list of potential interactors. Presence/absence of proteins was determined based on protein raw intensity (qualitative analysis). SUBAcon (consensus) location information was used to define subcellular targeting of protein partners (Hooper et al., 2014; Tanz et al., 2012).
Luzarowski M, Kosmacz M, Sokolowska E, Jasinska W, Willmitzer L, Veyel D, Skirycz A. Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases. J Exp Bot. 2017 68(13):3487-3499 PubMed: 28586477