Omics Assisted N-terminal Proteoform and Protein Expression Profiling On Methionine Aminopeptidase 1 (MetAP1) Deletion
2018; Elsevier BV; Volume: 17; Issue: 4 Linguagem: Inglês
10.1074/mcp.ra117.000360
ISSN1535-9484
AutoresVeronique Jonckheere, Daria Fijałkowska, Petra Van Damme,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoExcision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. Although MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression on MetAP1 deletion, MetAP2 was unable to restore processing of Met-Ser-, Met-Pro-, and Met-Ala- starting N termini as inferred from the iMet retention profiles observed, indicating a higher activity of MetAP1 over these N termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity. Excision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. Although MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression on MetAP1 deletion, MetAP2 was unable to restore processing of Met-Ser-, Met-Pro-, and Met-Ala- starting N termini as inferred from the iMet retention profiles observed, indicating a higher activity of MetAP1 over these N termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity. Protein biogenesis is one of the most fundamental and complex biological processes, with important implications for human health and disease. The concerted action of the ribosome and numerous ribosome-associated factors is essential to ensure proper initiation of translation, protein folding, processing and targeting. By the successive steps of ternary complex recruitment, ribosome scanning, start codon selection and ribosomal subunit joining, translation initiation acts as the gate-keeping step of translation. Here, protein biogenesis initiates with either an initiator N-formylmethionine (fMet) 1The abbreviations used are: fMet, N-formylmethionine; FDR, false discovery rate; iMet, initiator methionine; iBAQ, intensity based absolute quantification; LFQ, label free quantification; MetAP, methionine aminopeptidase; NAT, N-terminal acetyltransferase; NHEJ, non-homologous end-joining; NME, N-terminal methionine excision; Nt, N-terminal; PDF, peptide deformylase; RPKM, reads per kilobase of exon per million fragments; SCX, strong cation exchange; SREBP, sterol regulatory element binding protein; TIS, translation initiation site. 1The abbreviations used are: fMet, N-formylmethionine; FDR, false discovery rate; iMet, initiator methionine; iBAQ, intensity based absolute quantification; LFQ, label free quantification; MetAP, methionine aminopeptidase; NAT, N-terminal acetyltransferase; NHEJ, non-homologous end-joining; NME, N-terminal methionine excision; Nt, N-terminal; PDF, peptide deformylase; RPKM, reads per kilobase of exon per million fragments; SCX, strong cation exchange; SREBP, sterol regulatory element binding protein; TIS, translation initiation site. in case of bacterial and eukaryotic organellar protein synthesis, or an initiator methionine (iMet) in case of archaeal and eukaryotic cytosolic protein synthesis. Typically, for more than half of all nascent protein chains, the iMet is cotranslationally removed by the action of methionine aminopeptidases (MetAPs), a process referred to as N-terminal (Nt-) methionine excision (NME) (1.Giglione C. Boularot A. Meinnel T. Protein N-terminal methionine excision.Cell. Mol. Life Sci. 2004; 61: 1455-1474Crossref PubMed Scopus (239) Google Scholar). MetAPs represent a family of conserved and essential enzymes, as inferred from the lethality of the map null mutant in case of Salmonella typhimurium and Escherichia coli (2.Chang S.Y. McGary E.C. Chang S. Methionine aminopeptidase gene of Escherichia coli is essential for cell growth.J. Bacteriol. 1989; 171: 4071-4072Crossref PubMed Scopus (235) Google Scholar). Although typically only one MetAP gene is found in the genome of prokaryotes, eukaryotes express at least two types of cytosolic MetAPs belonging to a family of evolutionary conserved metalloproteases, being a type 1 and type 2 MetAP. Although MetAP2 by itself was shown to be essential for the development of specific tissues in multicellular organisms (3.Boxem M. Tsai C.W. Zhang Y. Saito R.M. Liu J.O. The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cell proliferation.FEBS Lett. 2004; 576: 245-250Crossref PubMed Scopus (26) Google Scholar), and MetAP1 appears to be vital for cell proliferation, a clear redundancy between MetAP1 and MetAP2 has been observed in Arabidopsis thaliana and Saccharomyces cerevisiae. Not surprisingly, double deletion mutants are nonviable, again pointing to their essential nature (4.Li X. Chang Y.H. Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 12357-12361Crossref PubMed Scopus (256) Google Scholar, 5.Ross S. Giglione C. Pierre M. Espagne C. Meinnel T. 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In case of eubacterial, mitochondrial and plastid NME, MetAPs remove the iMet of nascent polypeptide chains only after deformylation by the action of peptide deformylases (PDF) (8.Meinnel T. Mechulam Y. Blanquet S. Methionine as translation start signal: a review of the enzymes of the pathway in Escherichia coli.Biochimie. 1993; 75: 1061-1075Crossref PubMed Scopus (215) Google Scholar, 9.Solbiati J. Chapman-Smith A. Miller J.L. Miller C.G. Cronan Jr., J.E. Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal.J. Mol. Biol. 1999; 290: 607-614Crossref PubMed Scopus (127) Google Scholar). Further, the manifestation of (the extent of) iMet processing by MetAPs typically relates to the identity of the extreme N-terminal residues of the nascent polypeptide chain. More specifically, MetAPs only cleave iMet from nascent polypeptide chains in case of penultimate N-terminal residues (P1′) with (relatively) small and uncharged side chains (i.e. Gly, Ser, Ala, Pro, Cys, Thr, or Val) (10.Frottin F. Martinez A. Peynot P. Mitra S. Holz R.C. Giglione C. Meinnel T. The proteomics of N-terminal methionine cleavage.Mol. Cell. Proteomics. 2006; 5: 2336-2349Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The latter referred to as NME compatible residues. Furthermore, amino acid residues beyond P1′ may also contribute to the efficiency of NME (11.Xiao Q. Zhang F. Nacev B.A. Liu J.O. Pei D. Protein N-terminal processing: substrate specificity of Escherichia coli and human methionine aminopeptidases.Biochemistry. 2010; 49: 5588-5599Crossref PubMed Scopus (112) Google Scholar, 12.Arfin S.M. Bradshaw R.A. 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Interestingly and although still unclear, the need for having a MetAP of eubacterial and archaeal origin, being MetAP1 and MetAP2 respectively, might be attributed to (slight) differences in their substrate specificity profiles. Further, the N-terminal amino acid sequence is also the major feature determining whether or not a given protein is Nt-acetylated and by which Nt-acetyltransferase or NAT (16.Polevoda B. Sherman F. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins.J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (353) Google Scholar). As such, the interplay among other co-translational acting modifiers, such as the NATs responsible for the cotranslational Nt-acetylation of a large cohort of proteins in case of eukaryotes (17.Arnesen T. Van Damme P. Polevoda B. Helsens K. Evjenth R. Colaert N. Varhaug J.E. Vandekerckhove J. Lillehaug J.R. Sherman F. Gevaert K. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8157-8162Crossref PubMed Scopus (382) Google Scholar, 18.Brown J.L. Roberts W.K. Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are Nalpha-acetylated.J. Biol. Chem. 1976; 251: 1009-1014Abstract Full Text PDF PubMed Google Scholar, 19.Van Damme P. Hole K. Pimenta-Marques A. Helsens K. Vandekerckhove J. Martinho R.G. Gevaert K. Arnesen T. NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.PLoS Genet. 2011; 7: e1002169Crossref PubMed Scopus (126) Google Scholar), has also been implicated in determining MetAP susceptibility and thus (the extend of) iMet retention. More specifically we have previously shown Nt-acetylated iMet to be refractory toward the action of MetAPs (20.Van Damme P. Hole K. Gevaert K. Arnesen T. N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.Proteomics. 2015; 15: 2436-2446Crossref PubMed Scopus (39) Google Scholar). By having different processing enzymes operating at the ribosomal exit tunnel, each nascent protein may undergo several cotranslational modifications before being fully matured. Consequently, different N-terminal proteoforms might display characteristic functionalities, such as altered protein stabilities (21.Kim H.K. Kim R.R. Oh J.H. Cho H. Varshavsky A. Hwang C.S. The N-terminal methionine of cellular proteins as a degradation signal.Cell. 2014; 156: 158-169Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 22.Gawron D. Ndah E. Gevaert K. Van Damme P. Positional proteomics reveals differences in N-terminal proteoform stability.Mol. Syst. Biol. 2016; 12: 858Crossref PubMed Scopus (51) Google Scholar, 23.Varshavsky A. The N-end rule pathway and regulation by proteolysis.Protein Sci. 2011; 20: 1298-1345Crossref PubMed Scopus (478) Google Scholar) and subcellular localization. Consequently, Nt-modifications contribute to the increase of protein complexity observed. In this study, we studied the omics-wide effects of human MetAP1 deletion using a human haploid HAP1 MetAP1 knockout cell line model and compared it to general MetAP inhibition using Bengamide B, a marine-derived natural product with broad spectrum antitumor activity which functions as a nanomolar inhibitor of MetAPs 1 and 2 (24.Towbin H. Bair K.W. DeCaprio J.A. Eck M.J. Kim S. Kinder F.R. Morollo A. Mueller D.R. Schindler P. Song H.K. van Oostrum J. Versace R.W. Voshol H. Wood J. Zabludoff S. Phillips P.E. Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors.J. Biol. Chem. 2003; 278: 52964-52971Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The use of these different setups additionally enable discriminating more direct from indirect MetAP actions. By enriching for protein N termini using positional proteomics, we revealed the in vivo substrate profile of MetAP1 at the proteome-wide level, thereby aiding in elucidating the functional role of MetAPs. Furthermore, our proteome analysis showed that the increased expression of MetAP2 observed on MetAP1 deletion was unable to restore processing of MS-, MP- and MA- N termini, overall pointing to MetAP1 as the MetAP being exclusively or more active over these N termini and providing increasing knowledge on the in vivo substrate repertoire of MetAPs. Only in specific cases of which the iMet-processed and iMet unprocessed Nt-proteoforms could be observed, iMet processing affected the steady-state Nt-acetylation levels of Nt-proteoforms. Interestingly, changes in steady-state protein expression levels were typically explained at the transcript level and not related to protein N-terminal amino acid identities. More specifically, upon perturbation of MetAP activity, omics expression profiling revealed differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis. HAP1 control cells (HAP1 WT) and CRISPR/Cas9 edited human METAP1 knockout cells (HAP1 MetAP1 KO) containing a frameshift mutation in a coding exon of METAP1 (i.e. HAP1 cells carrying a 179bp insertion 'ATACAGAGTGACATAATGGACACTANAGACTCANAGTGGGGAGGGTAAGAGGGGGGCAAGGNATAAAAAACCACATATTGGGTACAATGTACACTACTCAGGTGACCGGTGCAGTAAAATCTCANACTTCNCTACTGTAGAATTCATCTATATGACCAAAAGCCATTTGTACCCCAAAA' in exon 4 of NM_015143 at chr4:99035439, leading to the corresponding MetAP1 genomic sequence of CAAAGACCAGAT(179 bp insertion)TATGCTGATCAT which was sequence verified by Sanger sequencing on PCR amplification from purified genomic DNA using the following primers; CTTTCTAAATACCCTGCAAAAGGGG (forward) and TCTTCAATGTTCTAATGGTGCTTG (reverse)) were obtained from Horizon Genomics GmbH (Vienna, Austria) and cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 50 units/ml penicillin and 50 μg/ml streptomycin (Invitrogen). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and passaged every 3–4 days. For the MetAP1/2 inhibition experiments, 0.1 μm f.c. Bengamide B (sc-397521A, Santa Cruz Biotechnology, Dallas, TX) was added to HAP1 cells cultured at about 80% of confluency. For proteome-analyses, cells were harvested after 24 h of treatment. Cold PBS washed HAP1 cells were lysed for 5 min on ice in RIPA buffer (50 mm Tris-HCl pH 8.0, 150 mm NaCl, 1% Nonidet P-40) with protease inhibitors added (Roche, Basel, Switzerland). For MetAP1 detection, HAP1 cells were lysed in urea lysis buffer (9 m urea, 50 mm NH4HCO3 (pH 7.9)) by three rounds of freeze-thaw lysis in liquid in N2. The urea lysates were sonicated (Branson probe sonifier output 4, 50% duty cycle, 3 × 30 s, 1 s pulses) followed by centrifugation for 10 min at 16,100 × g (4 °C). All lysates were cleared by centrifugation for 10 min at 16,100 g and protein concentration of the measured using the DC Protein Assay Kit (Bio-Rad, Munich, Germany). Sample loading buffer was added and equal amounts of protein (between 40 μg and 100 μg/sample) were separated by SDS-PAGE (1.0 mm thick 12% polyacrylamide Criterion Bis-Tris XT- gels, Bio-Rad) in MOPS buffer (Bio-Rad) at 150 V. Subsequently, proteins were transferred onto a PVDF membrane. Membranes were blocked for 30 min in a 1:1 Tris-buffered saline and 0.1% Tween-20 (TBS-T) Odyssey Blocking solution (cat n° 927–40003, LI-COR, Lincoln, NE) and probed by Western blotting. Following overnight incubation of primary antibody in TBS-T/Odyssey blocking buffer and three 10 min washes in TBS-T, membranes were incubated with secondary antibody for 1 h in TBS-T/Odyssey blocking buffer followed by 3 washes in TBS-T or TBS (last wash step). The following antibodies were used: rabbit anti-actin (A2066, Sigma, Saint-Louis, MO), mouse monoclonal unprocessed 14-3-3γ (iMet 14-3-3γ) (NB100–407, Novus Biologicals, Littleton, CO), rabbit polyclonal 14-3-3γ (orb128923, Biorbyt, Cambridge, UK), rabbit polyclonal anti-MetAP1 (NBP1–53088, Novus Biologicals), rabbit polyclonal anti-MetAP2 (orb33856, Biorbyt), rabbit anti-COTL (orb39513, Biorbyt), mouse monoclonal anti-cytokeratin 18 (ab668, Abcam, Cambridge, UK), rabbit anti-CRABP2 (orb32974, Biorbyt), rabbit anti-RL26A (ab59652, Abcam), rabbit anti-DUSP9 (orb338892, Biorbyt), rabbit anti-HMGCS (orb36825, Biorbyt), anti-mouse (IRDye 800 CW goat anti-mouse antibody IgG, LI-COR, cat n° 926–32210) and anti-rabbit (IRDye 800CW goat anti-rabbit IgG, LI-COR, cat n° 926–32211). The bands were visualized using an Odyssey infrared imaging system (LI-COR) and the intensity of bands assessed using the LICOR Odyssey software for Western blot image processing. Cell pellets of WT (HAP1 WT) and METAP1 KO (HAP1 MetAP1 KO) cells obtained from 3 independent cell cultures and containing approx. 10 × 106 cells per pellet, were collected and stored at −80 °C until further processing. HAP1 cell pellets resuspended in 400 μl Gu.HCl lysis buffer (4 m GdmCl, 50 mm NH4HCO3 (pH 7.9)) were lysed by three rounds of freeze-thaw lysis in liquid in N2. The lysates were sonicated (Branson probe sonifier output 4, 50% duty cycle, 3 × 30 s, 1 s pulses) followed by centrifugation for 10 min at 16,100 × g (4 °C), the supernatant removed and protein concentration determined by Bradford measurement according to the manufacturer's instructions. An aliquot equivalent of 200 μg (∼5 × 106 cells) was transferred to a clean tube, diluted to 1 mg/ml with lysis buffer, 2× diluted with HPLC grade water, and precipitated with 4× volumes of −20 °C acetone overnight. The precipitated protein material was recovered by centrifugation for 15 min at 3500 × g (4 °C), pellets washed twice with −20 °C 80% acetone, and air dried upside down for ∼10 min at RT or until no residual acetone odor remained. Pellets were resuspended in 200 μl TFE (2,2,2-trifluoroethanol) digestion buffer (10% TFE, 100 mm ammonium bicarbonate) with sonication until a homogenous suspension was reached. All samples were digested overnight at 37 °C using mass spec grade trypsin/Lys-C Mix (Promega, Madison, WI) (enzyme/substrate of 1:50 w/w) while mixing (550 rpm). Samples were acidified with TFA to a final concentration of 0.5%. Samples were cleared from insoluble particulates by centrifugation for 10 min at 16,100 × g (4 °C) and the supernatant transferred to clean tubes. Methionine oxidation was performed by the addition of H2O2 to reach a f.c. of 0.5% for 30′ at 30 °C. Solid phase extraction of peptides was performed using C18 reversed phase sorbent containing 100 μl pipette tips (Bond Elut OMIX 100 μl C18 tips (Agilent, Santa Clara, CA) according to the manufacturer's instructions. The pipette tip was conditioned by aspirating the maximum pipette tip volume of water:acetonitrile, 50:50 (v/v) and the solvent discarded. After equilibration of the tip by washing 3 times with the maximum pipette tip volume in 0.1% TFA in water, 100 μl of the acidified samples (∼200 μg) were dispensed and aspirated for 10 cycles for maximum binding efficiency. The tip was washed 3 times with the maximum pipette tip volume of 0.1% TFA in water:acetonitrile, 98:2 (v/v) and the bound peptides eluted in LC-MS/MS vials with the maximum pipette tip volume of 0.1% TFA in water:acetonitrile, 30:70 (v/v). The samples were vacuum-dried in a SpeedVac concentrator and re-dissolved in 20 μl of 2 mm tris(2-carboxyethyl)phosphine in 2% acetonitrile. Pellets of WT (HAP1 WT) and METAP1 KO (HAP1 MetAP1 KO) cells obtained from 3 independent cell cultures (i.e. corresponding cultures of the shotgun proteome analysis) and containing approx. 50 × 106 cells per pellet, were collected and stored at −80 °C until further processing. HAP1 cell pellets resuspended in 50 mm NH4HCO3 (pH 7.9) with protease inhibitors added (Roche) were lysed by three rounds of freeze-thaw lysis in liquid N2 and the lysates cleared by centrifugation for 10 min at 16,000 × g. Solid guanidinum hydrochloride was added to the supernatant to a final concentration of 4 m and the protein samples (equivalent of 2 mg) were reduced and S-alkylated. All primary protein amines were blocked using an N-hydroxysuccinimide ester of (stable isotopic encoded) acetate at the protein level (i.e. an NHS ester of 13C2D3 acetate) to enable the assignment and the calculation of the degree of in vivo Nt-acetylation events (25.Van Damme P. Arnesen T. Ruttens B. Gevaert K. In-gel N-acetylation for the quantification of the degree of protein in vivo N-terminal acetylation.Methods Mol. Biol. 2013; 981: 115-126Crossref PubMed Scopus (12) Google Scholar). All modified proteome samples were digested overnight at 37 °C using mass spec grade trypsin/Lys-C Mix (enzyme/substrate of 1:50 w/w) while mixing (550 rpm) and the resulting peptide mixtures vacuum dried. N-terminal peptide enrichment by SCX was performed as described previously (26.Staes A. Impens F. Van Damme P. Ruttens B. Goethals M. Demol H. Timmerman E. Vandekerckhove J. Gevaert K. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.Nat. Protocols. 2011; 6: 1130-1141Crossref PubMed Scopus (139) Google Scholar). The eluted fraction enriched for Nt-peptides were vacuum dried and re-dissolved in 1 ml of 0.5% TFA in 2% acetonitrile. Samples were cleared from insoluble particulates by centrifugation for 10′ at 16,100 g and 4 °C, and the supernatant transferred to clean tubes. Methionine oxidation was performed on 100 μl of acidified sample (∼200 μg) by the addition of H2O2 to reach a f.c. of 0.5% for 30′ at 30 °C. Solid phase extraction of peptides was performed using C18 reversed phase sorbent containing 100 μl pipette tips as described above. RNA was isolated from HAP1 cells using the TRIzol reagent (Invitrogen) according to manufacturer's instructions. RNA yields were determined using a NanoDrop spectrophotometer (Wilmington, Delaware) and RNA quality was assessed by Agilent Bioanalyzer RNA 6000 Nano Kit. Only samples with RIN values above 9 were accepted. Library generation including random fragmentation, cDNA synthesis and sequencing was performed at the VIB Nucleomics Core (www.nucleomics.be) using the TruSeq stranded total RNA sample preparation kit (Illumina, San Diego, CA) and including a Ribo-Zero (Illumina) depletion step as to remove ribosomal RNA from total RNA by the use of biotinylated Ribo-Zero oligos. Libraries were subjected to sequencing on a NextSeq 500 instrument (Illumina) to yield 75 bp single-end reads, resulting in ∼23–28 million 75 bp single end reads per sample. The 3′ adapter sequence (AGATCGGAAGAGCACAC) was trimmed using the fastx_clipper. Reads were pre-mapped onto small nuclear RNA, tRNA and rRNA. The remaining unmapped reads were then mapped onto the human GRCh37 reference genome (Ensembl annotation bundle 75) using STAR 2.4.0i allowing only unique mapping with a maximum of two mismatches. Separate BedGraph files were generated for the sense and antisense strand. Reads were counted across annotated genes using HTseq set to mode "union," feature type "exon" and minimum alignment quality of 10. For the MetAP1 KO cell line, two biological replicate samples were sequenced and for the WT cell line three. The unfiltered reads per kilobase of exon per million fragments (RPKM) values of protein-coding genes displayed a bimodal distribution, where approximately one third of genes had poor mRNA expression (i.e. genes with at least one read mapped but with RPKM values below 1), in line with previous reports, (27.Hebenstreit D. Fang M. Gu M. Charoensawan V. van Oudenaarden A. Teichmann S.A. RNA sequencing reveals two major classes of gene expression levels in metazoan cells.Mol. Syst. Biol. 2011; 7: 497Crossref PubMed Scopus (217) Google Scholar). Moreover, typically less than 2% of these poorly expressed genes have a corresponding protein expression detected using label-free shotgun proteomics. Therefore, and besides considering only unique reads from the mapping, only protein-coding genes with ≥ 1 RPKM in all biological replicates of both conditions were retained for differential expression analysis. Differential expression analysis was performed using the Bioconductor package DESeq2 (Stanford, CA) in the R statistical programming environment. To determine significantly regulated genes, an adjusted p value threshold of 0.01 was applied. The fold change calculations for differentially expressed genes was determined by DESeq2 and represent the log2 change in expression level (i.e. counts) for the MetAP1 KO versus the HAP1 control setup. RNA-seq sequencing data is accessible through GEO Series accession number GSE103405 in NCBI's Gene Expression Omnibus (28.Edgar R. Domrachev M. Lash A.E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.Nucleic Acids Res. 2002; 30: 207-210Crossref PubMed Scopus (8525) Google Scholar) with reviewer password: wpuxikegfnkjjkx. Peptides were separated by nano-LC and directly analyzed with a Q Exactive instrument (Thermo Scientific, Bremen, Germany) operating in MS/MS mode as described before (29.Stes E. Laga M. Walton A. Samyn N. Timmerman E. De Smet I. Goormachtig S. Gevaert K. A COFRADIC protocol to study protein ubiquitination.J. Proteome Res. 2014; 13: 3107-3113Crossref PubMed Scopus (45) Google Scholar). More specifically, all samples were analyzed via LC-MS/MS on an Ultimate 3000 RSLC nano LC (Thermo Fisher Scientific) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The sample mixture was first loaded on a trapping column (made in-house, 100 μm i.d. × 20 mm, 5 μm beads C18 Reprosil-HD, Dr. Maisch, Ammerbuch-Entringen, Germany). After flushing from the trapping column, the sample was loaded on an analytical column (made in-house, 75 μm i.d. × 150 mm, 3 μm beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with loading solvent (0.1% TFA in water) and separated with a linear gradient from 98% solvent A′ (0.1% formic acid in water) to 55% solvent B′ (0.1% formic acid in water/acetonitrile, 20:80 (v/v)) in 30 min at a flow rate of 300 nL/min. This was followed by a 5 min wash reaching 99% solvent B′. The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in each MS spectrum. The source voltage was 3.4 kV, and the capillary temperature was 275 °C. One MS1 scan (m/z 400–2000, AGC target 3 × 106 ions, maximum ion injection time 80 ms) acquired at a resolution of 70 000 (at 200 m/z) was followed by up to 10 tandem MS scans (resolution 17 500 at 200 m/z) of the most intense ions fulfilling predefined selection criteria (AGC target, 5 × 104 ions; maximum ion injection time, 60 ms; isolation window, 2 Da; fixed first mass, 140 m/z; spectrum data type, centroid; underfill ratio, 2%; intensity threshold, 1.7 × 104; exclusion of unassigned, 1, 5–8, >8 charged precursors; peptide match preferred; exclude isotopes, on; dynamic exclusion time, 20 s). The HCD collision energy was set to 25% normalized collision energy, and the polydimethylcyclosiloxane background ion at 445.120025 Da was used for internal calibration (lock mass). Raw data files were searched with MaxQuant (30.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar) using the Andromeda search engine (31.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3474) Google Scholar) (version 1.5.4.1) and MS/MS spectra searched against the Swiss-Prot database (taxonomy Homo sapiens; 20,195 entries; May 2016 version) for N-terminal proteome analyses, or a two amino acids N-terminally truncated version of the Swiss-Prot database was used to avoid contribution of N-terminal peptide abundances to the overall LFQ protein calculations in case of shotgun proteome analyses. In both cases potential contaminants present in the contaminants.fasta file that comes with MaxQuant were automatically added. A precursor mass tolerance was se
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