A review of COFRADIC techniques targeting protein N-terminal acetylation

Damme, Petra Van; Damme, Jozef Van; Demol, Hans; Staes, An; Vandekerckhove, Joël; Gevaert, Kris

doi:10.1186/1753-6561-3-s6-s6

Cited by 59 publications

(63 citation statements)

References 19 publications

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“…If needed, this labeling strategy allows for a straightforward calculation of the extent of N-terminal acetylation (29). After tryptic digestion, all protein N-terminal peptides will thus be blocked, whereas internal peptides acquired a newly generated primary ␣-amine, a property exploited to isolate N-terminal peptides from internal peptides in a diagonal chromatography setup that follows strong cation exchange enrichment at low pH (25).…”

Section: Resultsmentioning

confidence: 99%

Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

Menschaert

Criekinge

Notelaers

et al. 2013

Molecular & Cellular Proteomics

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155

143

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An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both Swiss-Prot-and RIBO-seq-derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database, we performed two alternative MS-based proteomic strategies as follows: (i) a regular shotgun proteomic and (ii) an N-terminal combined fractional diagonal chromatography (COFRADIC) approach. Although the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq-derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate 2.5% (e.g. new protein products, new protein splice variants, single nucleotide polymorphism variant proteins, and N-terminally extended forms of known proteins) as compared with only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated upstream ORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA sequencing and especially custom-tailored RIBO-seq will become indispensable in the MS-based protein or peptide identification process. The underlying mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000124. Molecular & Cellular

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Section: Resultsmentioning

confidence: 99%

Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

Menschaert

Criekinge

Notelaers

et al. 2013

Molecular & Cellular Proteomics

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155

143

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“…To elucidate the in vivo Nt-acetylomes of yNatB and hNatB and to delineate potential differences in their in vivo substrate specificity profiles in yeast, quantitative N-terminal COFRADIC analyses were performed (1,25), and the Nt-acetylation states of wild-type, yNatB-Δ and y[hNatB] yeast proteomes were compared. To distinguish between in vivo Nt-acetylated and non-Nt-acetylated N termini, we used chemical in vitro 13 C 2 D 3 -acetylation, which introduces a 5-Da mass spacing between the non-Nt-acetylated and Nt-acetylated N-terminal peptide form and thereby allows the calculation of the extent of Nt-acetylation (1,26). Overall, in the three yeast strains analyzed, 1,623 unique yeast N termini originating from 1,321 yeast proteins were identified (Table S1).…”

Section: Resultsmentioning

confidence: 99%

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

Damme

Lasa²,

Polevoda

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln-N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

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“…Recently, this altered biophysical property was also exploited to enrich for protein N-termini using low pH strong cation exchange (SCX) chromatography (24,39). As an example, SCX prefractionation combined with N-terminal combined fractional diagonal chromatography, a targeted proteomics technology negatively selecting for protein N-terminal peptides, stable isotope labeling of amino acids in cell culture, and amino-directed modifiers (40), was used to study the in vivo substrate repertoires of human as well as yeast NatA (4). Nevertheless, the various methods reported today to study in detail N ␣ -terminal acetylation and thus the specificities of different NATs make use of a limited and therefore somewhat biased set of synthesized peptide substrates and comprise the rather laborious detection of radioactive acetylated products as well as enzyme-coupled methods quantifying acetylCoA conversion.…”

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confidence: 99%

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

Damme

Evjenth

Foyn

et al. 2011

Molecular & Cellular Proteomics

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219

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The impact of N ␣ -terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N ␣ -terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N ␣ -acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are ␥-and ␤-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both ␥-and ␤-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore,

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A review of COFRADIC techniques targeting protein N-terminal acetylation

Cited by 59 publications

References 19 publications

Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

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