Proteome-derived Peptide Libraries Research Articles

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Proteome-Derived Peptide Libraries for Deep Specificity Profiling of N-terminal Modification Reagents.

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal ⍺-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates through tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome-derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries.

Profiling Sequence Specificity of Proteolytic Activities Using Proteome-Derived Peptide Libraries.

Substrate sequence specificity is a fundamental characteristic of proteolytic enzymes. Hundreds of proteases are encoded in plant genomes, but the vast majority of them have not been characterized and their distinct specificity remains largely unknown. Here we present our current protocol for profiling sequence specificity of plant proteases using Proteomic Identification of Cleavage Sites (PICS). This simple, cost-effective protocol is suited for detailed, time-resolved specificity profiling of purified or enriched proteases. The isolated active protease or fraction with enriched protease activity together with a suitable control are incubated with split aliquots of proteome-derived peptide libraries, followed by identification of specifically cleaved peptides using quantitative mass spectrometry. Detailed specificity profiles are obtained by alignment of many individual cleavage sites. The chapter covers preparation of complementary peptide libraries from heterologous sources, the cleavage assay itself, as well as mass spectrometry data analysis.

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches.

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, includingneo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Specificity profiling of human trypsin-isoenzymes.

In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1' position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1' and P2' preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2' is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.

Protease Specificity Profiling in a Pipet Tip Using "Charge-Synchronized" Proteome-Derived Peptide Libraries.

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method's versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme–substrate specificity limits its utility. Here, we present an approach for comprehensive characterization of peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme–substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. These studies provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

The cleavage preferences of Kallikrein-related peptidase 7 (KLK7) have previously been delineated using synthetic peptide libraries of fixed length, or single protein chains and have suggested that KLK7 exerts a chymotryptic-like cleavage preference. Due to the short length of the peptides utilised, only a limited number of subsites have however been assessed. To determine the subsite preferences of KLK7 in a global setting, we used a mass spectrometry (MS)-based in-depth proteomics approach that utilises human proteome-derived peptide libraries of varying length, termed Proteomic Identification of protease Cleavage Sites (PICS). Consistent with previous findings, KLK7 was found to exert chymotryptic-like cleavage preferences. KLK7 subsite preferences were also characterised in the P2-P2′ region, demonstrating a preference for hydrophobic residues in the non-prime and hydrophilic residues in the prime subsites. Interestingly, single catalytic triad mutant KLK7 (mKLK7; S195A) also showed residual catalytic activity (kcat/KM = 7.93 × 102 s−1M−1). Catalytic inactivity of KLK7 was however achieved by additional mutation in this region (D102N). In addition to characterising the cleavage preferences of KLK7, our data thereby also suggests that the use of double catalytic triad mutants should be employed as more appropriate negative controls in future investigations of KLK7, especially when highly sensitive MS-based approaches are employed.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B

The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics.

Biochemical profiling of active site specificity is a crucial step to characterize proteases, which play key roles in health and disease. Here, we present a protocol using proteome-derived peptide libraries in combination with quantitative proteomics to simultaneously identify cleavage motifs N- and C-terminal to the scissile peptide bond. First, bacterial or eukaryotic cell lysate is used to generate peptide libraries. Without further chemical modification, peptide libraries are then split into control and treated (incubate with active protease) aliquots. Control and treated libraries are stable isotope-labeled, mixed, and analyzed by liquid chromatography-tandem mass spectrometry. Enriched, semi-specific peptides represent the cleavage products of the test protease and the entire peptide sequence that encompasses the scissile peptide bond is reconstructed bioinformatically. The method is fast, cost-effective, and suited for proteases with narrow or loose specificity.

Multiplexed Protease Specificity Profiling Using Isobaric Labeling.

The determination of a protease's cleavage site specificity is one of the major goals in degradomics. In the last years, the use of proteome-derived peptide libraries and liquid chromatography-mass spectrometry (LC-MS) in a proteomic identification of protease cleavage sites (PICS) experiment became popular for that purpose.In this chapter, we offer a step-by-step protocol for the execution of a quantitative proteomic identification of protease cleavage sites (Q-PICS) experiment, which enables the relative quantification of proteolytic events by isobaric labeling, e.g., with tandem mass tags (TMT). In this way, the cleavage site specificity and activity of a protease can be compared under different reaction conditions (e.g., buffer, pH, temperature, inhibitor). Multiplexing can further be used to analyze replicate experiments in parallel, decreasing instrument times and work effort significantly, or to perform internal controls.

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity

The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.

Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.

Proteome-derived peptide library for the elucidation of the cleavage specificity of HF3, a snake venom metalloproteinase.

The Proteomic Identification of Cleavage Sites (PICS) approach was employed for profiling the substrate specificity of HF3, a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca. A tryptic peptide library from human plasma was subject to HF3 cleavage and amino acid occurrence for P6 to P6' sites was mapped. 71 cleavage sites were detected and revealed a clear preference for leucine at P1' position, followed by hydrophobic residues in P2'. PICS confirmed existing data on prime site specificity of SVMPs.

Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations

Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome (“secretome”), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) β, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFβ. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By “terminal amine labeling of substrates” (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor–stroma interaction to FAPα.

Protease subsite profiling with proteome-derived peptide libraries (PICS)

Protease subsite profiling with proteome-derived peptide libraries (PICS)

Identification of the PLK2-Dependent Phosphopeptidome by Quantitative Proteomics

Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.