Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

NatB-Mediated N-Terminal Acetylation Affects Growth and Biotic Stress Responses.

N∝-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis (Arabidopsis thaliana) NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, and naa25-1) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement naa20-1 Quantitative N-terminomics of approximately 1000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N termini starting with the initiator Met followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity toward osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors.

NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern.

An organism's entire protein modification repertoire has yet to be comprehensively mapped. N-myristoylation (MYR) is a crucial eukaryotic N-terminal protein modification. Here we mapped complete Homo sapiens and Arabidopsis thaliana myristoylomes. The crystal structures of human modifier NMT1 complexed with reactive and nonreactive target-mimicking peptide ligands revealed unexpected binding clefts and a modifier recognition pattern. This information allowed integrated mapping of myristoylomes using peptide macroarrays, dedicated prediction algorithms, and in vivo mass spectrometry. Global MYR profiling at the genomic scale identified over a thousand novel, heterogeneous targets in both organisms. Surprisingly, MYR involved a non-negligible set of overlapping targets with N-acetylation, and the sequence signature marks for a third proximal acylation-S-palmitoylation-were genomically imprinted, allowing recognition of sequences exhibiting both acylations. Together, the data extend the N-end rule concept for Gly-starting proteins to subcellular compartmentalization and reveal the main neighbors influencing protein modification profiles and consequent cell fate.

Two N-terminal acetyltransferases antagonistically regulate the stability of a nod-like receptor in Arabidopsis.

Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.

EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples.

BACKGROUND: Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones. RESULTS: Based on large scale proteomics analyses using the SILProNAQ methodology, tandem mass spectrometry favoured the characterization of thousands of peptides. Data processing using the EnCOUNTer tool provided an efficient and rapid way to extract the most reliable mature protein N-termini. Selected peptides were subjected to N-terminus acetylation yield determination. In an A. thaliana cell lysate, 1232 distinct proteotypic N-termini were characterized of which 648 were located at the predicted protein N-terminus (position 1/2) and 584 were located further downstream (starting at position > 2). A large number of these N-termini were associated with various well-defined maturation processes occurring on organelle-targeted proteins (mitochondria, chloroplast and peroxisome), secreted proteins or membrane-targeted proteins. It was also possible to highlight some protein alternative starts, splicing variants or erroneous protein sequence predictions. CONCLUSIONS: The EnCOUNTer tool provides a unique way to extract accurately the most relevant mature proteins N-terminal peptides from large scale experimental datasets. Such data processing allows the identification of the exact N-terminus position and the associated acetylation yield.

RPM-1 uses both ubiquitin ligase and phosphatase-based mechanisms to regulate DLK-1 during neuronal development.

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.

Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition.

A proteome wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of actinonin, an antibiotic specifically inhibiting peptide deformylase (PDF). A strategy and tool suite (SILProNaQ) was employed to provide large-scale quantitation of N-terminal modifications. In control conditions, more than 1000 unique N-termini were identified with 56% showing initiator methionine removal. Additional modifications corresponded to partial or complete Nα-acetylation (10%) and N-formyl retention (5%). Among the proteins undergoing these N-terminal modifications, 140 unique N-termini from translocated membrane proteins were highlighted. The very early time-course impact of actinonin was followed after addition of bacteriostatic concentrations of the drug. Under these conditions, 26% of all proteins did not undergo deformylation any longer after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation ratio measured on individual proteins increased with the same trend. Upon early PDF inhibition, two major categories of proteins retained their N-formyl group: a large number of inner membrane proteins and many proteins involved in protein synthesis including factors assisting the nascent chains in early cotranslational events. All MS data have been deposited in the ProteomeXchange with identifiers PXD001979, PXD002012 and PXD001983 (http://proteomecentral.proteomexchange.org/dataset/PXD001979, http://proteomecentral.proteomexchange.org/dataset/PXD002012 and http://proteomecentral.proteomexchange.org/dataset/PXD001983).

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

Protein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947).

Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features.

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Identification of protein interfaces between α-synuclein, the principal component of Lewy bodies in Parkinson disease, and the molecular chaperones human Hsc70 and the yeast Ssa1p.

Fibrillar α-synuclein (α-Syn) is the principal component of Lewy bodies, which are evident in individuals affected by Parkinson disease (PD). This neuropathologic form of α-Syn plays a central role in PD progression as it has been shown to propagate between neurons. Tools that interfere with α-Syn assembly or change the physicochemical properties of the fibrils have potential therapeutic properties as they may be sufficient to interfere with and/or halt cell-to-cell transmission and the systematic spread of α-Syn assemblies within the central nervous system. Vertebrate molecular chaperones from the constitutive/heat-inducible heat shock protein 70 (Hsc/p70) family have been shown to hinder the assembly of soluble α-Syn into fibrils and to bind to the fibrils and very significantly reduce their toxicity. To understand how Hsc70 family members sequester soluble α-Syn, we set up experiments to identify the molecular chaperone-α-Syn surface interfaces. We cross-linked human Hsc70 and its yeast homologue Ssa1p and α-Syn using a chemical cross-linker and mapped the Hsc70- and Ssa1p-α-Syn interface. We show that the client binding domain of Hsc70 and Ssa1p binds two regions within α-Syn similar to a tweezer, with the first spanning residues 10-45 and the second spanning residues 97-102. Our findings define what is necessary and sufficient for engineering Hsc70- and Ssa1p-derived polypeptide with minichaperone properties with a potential as therapeutic agents in Parkinson disease through their ability to affect α-Syn assembly and/or toxicity.

Influence of various endogenous and artefact modifications on large-scale proteomics analysis.

RATIONALE: Some large-scale proteomics studies in which strong cation exchange chromatography has been applied are used to determine proteomes and post-translational modification dynamics. Although such datasets favour the characterisation of thousands of modified peptides, e.g., phosphorylated and N-α-acetylated, a large fraction of the acquired spectra remain unexplained by standard proteomics approaches. Thus, advanced data processing allows characterisation of a significant part of these unassigned spectra. METHODS: Our recent investigation of the N-α-acetylation status of plant proteins gave a dataset of choice to investigate further the in-depth characterisation of peptide modifications using Mascot tools associated with relevant validation processes. Such an approach allows to target frequently occurring modifications such as methionine oxidation, phosphorylation or N-α-acetylation, but also the less usual peptide cationisation. Finally, this dataset offers the unique opportunity to determine the overall influence of some of these modifications on the identification score. RESULTS: Although methionine oxidation has no influence and tends to favour the characterisation of protein N-terminal peptides, peptide alkalinisation shows an adverse effect on peptide average score. Nevertheless, peptide cationisation appears to favour the characterisation of protein C-terminal peptides with a limited to no direct influence on the identification score. Unexpectedly, our investigation reveals the unfortunate combination of the molecular weight of N-α-acetylation and potassium cation that mimics the mass increment of a phosphorylation group. CONCLUSIONS: Since these characterisations rely upon computational treatment associated with statistical validation approaches such as 'False discovery rates' calculation or post-translational modification position validation, our investigation highlights the limitation of such treatment which is biased by the initial searched hypotheses.

HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.

Dynamics of post-translational modifications and protein stability in the stroma of Chlamydomonas reinhardtii chloroplasts.

The proteome of any system is a dynamic entity dependent on the intracellular concentration of the entire set of expressed proteins. In turn, this whole protein concentration will be reliant on the stability/turnover of each protein as dictated by their relative rates of synthesis and degradation. In this study, we have investigated the dynamics of the stromal proteome in the model organism Chlamydomonas reinhardtii by characterizing the half-life of the whole set of proteins. 2-DE stromal proteins profiling was set up and coupled with MS analyses. These identifications featuring an average of 26% sequence coverage and eight non-redundant peptides per protein have been obtained for 600 independent samples related to 253 distinct spots. An interactive map of the global stromal proteome, of 274 distinct protein variants is now available on-line at http://www.isv.cnrs-gif.fr/gel2dv2/. N-α-terminal-Acetylation (NTA) was noticed to be the most frequently detectable post-translational modification, and new experimental data related to the chloroplastic transit peptide cleavage site was obtained. Using this data set supplemented with series of pulse-chase experiments, elements directing the relationship between half-life and N-termini were analyzed. Positive correlation between NTA and protein half-life suggests that NTA could contribute to protein stabilization in the stroma.

C. elegans RPM-1 regulates axon termination and synaptogenesis through the Rab GEF GLO-4 and the Rab GTPase GLO-1.

C. elegans RPM-1 (for Regulator of Presynaptic Morphology) is a member of a conserved protein family that includes Drosophila Highwire and mammalian Pam and Phr1. These are large proteins recently shown to regulate synaptogenesis through E3 ubiquitin ligase activities. Here, we report the identification of an RCC1-like guanine nucleotide exchange factor, GLO-4, from mass spectrometry analysis of RPM-1-associated proteins. GLO-4 colocalizes with RPM-1 at presynaptic terminals. Loss of function in glo-4 or in its target Rab GTPase, glo-1, causes neuronal defects resembling those in rpm-1 mutants. We show that the glo pathway functions downstream of rpm-1 and acts in parallel to fsn-1, a partner of RPM-1 E3 ligase function. We find that late endosomes are specifically disorganized at the presynaptic terminals of glo-4 mutants. Our data suggest that RPM-1 positively regulates a Rab GTPase pathway to promote vesicular trafficking via late endosomes.

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability.

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.

Precursor ion scans for the targeted detection of stable-isotope-labeled peptides.

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique.

SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation.

Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.