WO2015030585A2 - Procédés pour détecter des lysines ayant subi une modification post-traductionnelle dans un polypeptide - Google Patents

Procédés pour détecter des lysines ayant subi une modification post-traductionnelle dans un polypeptide Download PDF

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WO2015030585A2
WO2015030585A2 PCT/NL2014/050581 NL2014050581W WO2015030585A2 WO 2015030585 A2 WO2015030585 A2 WO 2015030585A2 NL 2014050581 W NL2014050581 W NL 2014050581W WO 2015030585 A2 WO2015030585 A2 WO 2015030585A2
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lysine
post
sample
protein
sumo
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WO2015030585A3 (fr
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Alfredus Cornelis Otto VERTEGAAL
Ivo Alexander HENDRIKS
Matthias Mann
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Academisch Ziekenhuis Leiden H.O.D.N. Lumc
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36—Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to methods for detecting a post-translationally modified lysines in a polypeptide.
  • a specific type of post-translational lysine modification can be mapped at the level of the proteome.
  • One aspect of the disclosure provides a method for detecting a post-translationally modified lysine in a polypeptide in a sample, the method comprising: a) chemically modifying essentially all lysines having a free epsilon amino group in said sample, thereby rendering the peptide bond between said lysines and an adjacent amino acid resistant to proteolytic cleavage,
  • the lysines of step a) having a free epsilon amino group are chemically modified by acetylation and the single type of PTM from step b) is selected from alkylation, ubiquitination, methylation, and SUMOylation.
  • said single type of PTM is SUMOylation, and said PTM is removed by an Ulp enzyme, preferably selected from Ulp l, Ulp2, SENP1, SENP2, SENP3, SENP5, SENP6 or SENP7.
  • said protease is selected from trypsin and Lys-C.
  • the lysine at the C-terminus of a peptide from step d) indicates the site of a post-translational lysine modification.
  • said protease is selected from Lys-N.
  • a lysine at the N- terminus of a peptide from step d) indicates the site of a post- translational lysine modification.
  • the peptide is detected using mass spectrometry.
  • the method further comprises enriching said sample for polypeptides comprising said single type PTM .
  • said enrichment step comprises contacting said sample with a binding body molecule specific for said PTM .
  • the method further comprises enriching for polypeptides produced in step b) in which a single type of post-translational modification (PTM) from said post- translationally modified lysine is removed, produced in step d) having a lysine with a free amine group.
  • PTM post-translational modification
  • the method is for comparing the post-translational modification state of a lysine of a polypeptide between said first sample and a second sample, preferably wherein said first sample is subjected to a physical, mechanical, or chemical treatment (e.g., treatment to drugs/compounds (e.g., growth factors, hormones, small chemical molecules), temperature change, osmotic shock, pH change, light etc.) or wherein said first sample and said second sample differ in age, disease state, or source material , the method comprising:
  • the method is for determining whether a compound modulates the post- translational lysine modification state of one or more polypeptides, said method comprising contacting a first sample with a compound, and further comprising the steps:
  • the method is for determining whether a specific condition or disorder is associated with altered lysine post-translational modification, comprising providing a first sample obtained from an individual having a specific condition or disorder, or from an animal or cell culture model system for said specific condition or disorder, providing a second sample not affected with said condition or disorder, said method comprising:
  • the lysine with a free epsilon amino group represents a previously modified lysine.
  • the proteolytic cleavage referred to is preferably a proteolytic cleavage by a protease that cleaves a peptide bond between a lysine with a free epsilon amino group and an adjacent amino acid.
  • the invention further provides a method for detecting whether a protein contains a post-translational modification said method comprising
  • binding molecules binds said protein depending on the presence or absence of the post- translation modification and the other binding molecule binds said protein
  • the first and/or said second binding molecule is preferably an antibody.
  • Said post-translational modification is preferably a proteinaceous translational modification.
  • Said surfactant is preferably an anionic surfactant.
  • Said surfactant is preferably an amphiphilic organosulphate.
  • the surfactant is sodium dodecyl sulfate (SDS).
  • Said surfactant is preferably added in step ii, in an amount in excess of 0.5% weight/weight (of the total weight of the sample + surfactant).
  • Said capture protein is preferably added in an amount sufficient to reduce the amount of free surfactant to 0.1% or less (of the total weight of the sample + surfactant + capture protein).
  • Said first binding molecule preferably binds said protein depending on the presence or absence of the post- translation modification.
  • Said first binding molecule preferably binds an antigen on the proteinaceous post-translation modification.
  • Said first binding molecule is preferably linked to a solid surface.
  • said proteinaceous post-translational modification preferably comprises a ubiquitin-like protein, preferably a SUMO protein or ubiquitin protein or other ubiquitin family member including but not limited to Nedd8, Fubi, Hub l, ISG15, FAT 10, URM1, UFMl, SAMPl, SAMP2 or PUP.
  • Said proteinaceous post-translational modification in one embodiment comprises a tagged SUMO protein or tagged ubiquitin protein.
  • Said capture protein preferably comprises a serum albumin, preferably bovine serum albumin.
  • Said capture protein is preferably added in amount of at least 20% and preferably at least 40%, more preferably at least 60%, more preferably at least 100% relative to the amount of surfactant present in the sample of step iii (% given in weight/weight capture protein/surfactant).
  • Said sample is preferably a tissue sample.
  • the invention further provides a method for determining whether a compound affects the post- translation modification of a protein, said method characterized in that said protein is detected by a method for detecting whether a protein contains a (proteinaceous) post- translational modification according to the invention.
  • FIG. 1 An exemplary embodiment of a new method to detect lysine post- translational modifications.
  • the method starts with a polypeptide or mixture of polypeptides including one or more polypeptides that are post-translationally modified on one or more lysine residues.
  • all non-modified lysines are chemically blocked.
  • the lysine modification of interest is removed, in this example using a specific protease, thereby generating one or more unique non- modified lysines in these polypeptides.
  • a protease such as trypsin, Lys-C or Lys-N is used that specifically cleaves at the previously modified lysine to generate unique reporter ions representing the previously modified lysine.
  • Figure 2 Ni-NTA enrichment of Hisl0-SUMO2-AllKR-Q87R-conjugated proteins from HeLa cells was performed. One pool of cells was acetylated on-beads using sulfosuccinimidyl-acetate, while the other pool was mock treated. Following elution from the beads, the proteins were mock treated or processed with SUMO-specific protease SENP2.
  • FIG. 3 Ubiquitin-conjugated proteins within a total lysate were completely denatured under harsh conditions. Following renaturation, ubiquitin- specific protease USP2 is still able to recognize and remove ubiquitin from its target proteins.
  • HeLa cells were lysed in 8M urea, 50 sodium phosphate pH 8.5 supplemented with 70 mM chloroacetamide. The lysate was sonicated and cleared by centrifugation, and diluted to 1M urea by addition of 50 mM phosphate pH 8.5. The lysate was split in two fractions, and one fraction was mock treated whereas the other fraction was processed overnight with ubiquitin specific protease USP2. Protein samples were size-separated by SDS-PAGE, transferred to a nitrocellulose membrane, probed using a ubiquitin- specific antibody, and visualized through chemiluminescence.
  • Figure 4 Detecting SUMOylated Cockayne syndrome protein CSB using a sandwich ELISA approach.
  • HeLa cells stably expressing low levels of Flag-SUMO-2 were established using a lentiviral approach and analysed by immunoblotting using antibodies directed against
  • SUMO-2 conjugates were purified from HeLa cells stably expressing His6-SUMO-2 either non-exposed, or exposed to 20 J/m2 UV-C and harvested 1 hour after treatment. His6-SUMO-2 conjugates were purified by Immobilized Metal Affinity Chromatography (IMAC) and resulting samples were size- separated by SDS-PAGE and analysed by immunoblotting using an antibody directed against CSB.
  • IMAC Immobilized Metal Affinity Chromatography
  • SENP2 is a SUMO-specific protease able to cleave SUMO-2/3 from endogenous proteins under very stringent buffer conditions.
  • HeLa cells were lysed in 8 M urea, homogenized, and subsequently diluted to indicated lower concentrations of urea. Lysates were treated with SENPl, SENP2, or mock treated. After protease treatment, lysates were size-separated by SDS-PAGE, transferred to membranes, probed using a SUMO-2/3 antibody, and visualized using chemiluminescence .
  • RanGAPl is indicated with an arrow.
  • SENP2 is able to remove acetylated SUMO from acetylated proteins after sulfosuccinimidyl-acetate (SNHSA) treatment.
  • HeLa cells were lysed in 8 M urea, homogenized, and subsequently treated with 10 mM SNHSA or mock treated. Next, both lysine-blocked and control samples were treated with either trypsin or Lys-C, or mock treated. All samples were size-separated by SDS-PAGE, and total protein content was visualized using Coomassie.
  • HeLa cells stably expressing lysine -deficient HislO-tagged SUMO-2 were lysed and homogenized, and subsequently SUMOylated proteins were enriched by Ni-NTA pulldown.
  • SUMOylated proteins were treated on-beads with SNHSA or mock treated, prior to elution. After elution, proteins were either treated with SENP2 or mock treated. Finally, samples were digested using Lys-C or mock digested. All samples were size-separated using SDS-PAGE, transferred to membranes, probed using a SUMO-2/3 antibody, and visualized using chemiluminescence.
  • FIG. 7 A schematic overview of the PRISM double purification strategy.
  • A) 1. Cells are lysed under denaturing conditions, inactivating endogenous proteases. 2. SUMOylated proteins are pre-enriched using, in this case, Ni-NTA pulldown to capture the histidine tag. 3. SUMOylated proteins are acetylated on-beads using SNHSA under highly denaturing conditions, enabling efficient blocking of lysines. Excess chemical is washed away, and proteins are eluted using a buffer compatible with a second chemical labeling step. 4. Following elution, lysine-blocked
  • SUMOylated proteins are treated with SENP2, efficiently removing SUMO-2 and freeing up the lysines SUMO-2 was conjugated to. 5.
  • Freed lysines are biotinylated using SNHSSSB. 6.
  • Biotinylated SUMO target proteins are enriched using avidin pulldown, and either specifically eluted using a reducing (DTT) elution buffer, or totally eluted using LDS elution buffer.
  • DTT reducing
  • SUMO target proteins may be analyzed by various biochemical methods, such as immunoblotting.
  • SUMOylated proteins were purified from HeLa cells expressing lysine -deficient His-tagged SUMO-2, using PRISM as described in section A.
  • the assay was performed with and without the use of SNHSA to block lysines, and eluted proteins were either treated with SENP2 or mock treated.
  • Samples were size- separated using SDS-PAGE, transferred to membranes, probed using a TRIM33 antibody, and visualized using chemiluminescence.
  • Non-blocked TRIM33 eluted after Ni-NTA pulldown, Step 2 is indicated.
  • Lysine-blocked TRIM33 eluted after Ni-NTA pulldown, Step 3 is indicated.
  • Lysine-blocked TRIM33 that was successfully deSUMOylated by SENP2, Step 4, is indicated.
  • Cells are lysed under denaturing conditions, completely inactivating endogenous proteases.
  • SUMOylated proteins are pre-enriched using, in this case, Ni-NTA pulldown to capture the histidine tag.
  • SUMOylated proteins are acetylated on-beads using SNHSA under highly denaturing conditions, enabling efficient blocking of lysines. Excess chemical is washed away, and proteins are eluted using a buffer compatible with a second chemical labeling step. 4. Following elution, proteins are concentrated over a 100 kDa filter under denaturing conditions, specifically removing free SUMO but retaining SUMO-modified proteins. 5.
  • Concentrated lysine-blocked SUMOylated proteins are treated with SENP2, efficiently removing SUMO-2 and freeing up the lysines SUMO-2 was conjugated to. 6.
  • DeSUMOylated proteins are concentrated over a 100 kDa filter under denaturing conditions, removing SUMO cleaved off by SENP2 while retaining proteins previously SUMO-modified.
  • Concentrated SUMO target proteins are trypsinized, with trypsin only able to cleave arginines because all lysines in proteins are blocked, with the exception of the lysines that were freed by SENP2.
  • two reporter peptides are generated per site, either ending in a lysine or preceded by a lysine.
  • Peptides are analyzed using high-resolution mass
  • SUMOylated proteins were purified from medium and heavy SILAC labeled HeLa cells stably expressing His-tagged SUMO-2, using PRISM as described in section A. During various steps of the purification samples were taken for diagnostic purposes. Samples were size -separated using SDS-PAGE, transferred to membranes, probed using a SUMO-2/3 antibody, and visualized using chemiluminescence. Samples are indicated by the corresponding step number from section A.
  • Term enrichment analysis comparing all SUMO target proteins identified by PRISM to the human proteome. Gene Ontology Molecular Functions terms were used to find statistical enrichments within the SUMO target protein dataset. Term enrichment score is a composite score based on enrichment over randomly expected and the negative logarithm of the false discovery rate. All listed terms are significant with p « 0.02.
  • Figure 10 A comparison of PRISM-identified SUMOylation sites and target proteins to other SUMOylation studies.
  • the methods described herein allow for the detection of lysine residues which have been post-translationally modified.
  • the lysines may be found in any polypeptide whether naturally or non-naturally occurring.
  • the polypeptides may be synthetically produced, or produced either in vitro or in vivo.
  • Andersen et al. (Molecular Cell 43, 834-842 2011) describes a method based on the biotin-switch methodology for identifying deacetylase substrates. The method results in peptides in which the previously acetylated lysines are now alkylated. The identity of the resulting peptides is determined using mass spectrometry.
  • the present invention converts the previously modified lysine that was resistant to protease cleavage (e.g., by Lys-C, Lys-N and trypsin) to a non- modified lysine that is subsequently cleaved by a protease (e.g., trypsin, Lys-C or Lys- N) and thereby generates two unique reporter ions, one being the peptide that is cleaved at the previously modified lysine and one being the directly adjacent peptide that is also unique due to the presence of the cleavage site.
  • lysine protection is performed under denaturing conditions, which is useful for achieving complete protection.
  • lysine blockages by Andersen is performed under non- denaturing conditions, which is more likely to result in partial blockage of lysines due to 3D folding of proteins.
  • the method of the invention does not require a second chemical modification step as described in Andersen et al.
  • the present invention avoids partial elution of biotinylated peptides from Avidin or Streptavidin resins that could limit the yield of the purified material.
  • the present methods result in the generation of peptides which have been cleaved either N- or C-terminal to the previously post-translationally modified lysine. This results in the generation of two reporter ions for mass spectrometry, improving the identification rate of modifications.
  • the identification of the relevant peptides by mass spectrometry is easier compared to the method described by Andersen et al., since the previously modified lysines are converted to native lysines.
  • the disclosure provides a method for detecting a post-translationally modified lysine in a polypeptide in a sample, the method comprising:
  • the detection of a post-translationally modified lysine in a polypeptide in a sample also encompasses the post-translational modification state of one or more polypeptides in a sample and the determination of the lysine post-translational modification state of a sample , or rather, to identify at a systems level the PTM lysine state of a sample.
  • the methods can detect one or more specific post-translationally modified lysines in one or more polypeptides, preferably in a single polypeptide.
  • peptides comprising a previously modified lysine residue are detected.
  • the methods disclosed herein also allow for the comparison of the post-translational modification state of a lysine between samples.
  • the two samples may differ, e.g., in age, disease state, source material (e.g. normal versus tumor tissue), prior treatment (e.g., any mechanical, physical or chemical treatment such as treatment with drugs/compounds (e.g., growth factors, hormones, small chemical molecules), temperature change, osmotic shock, pH change, light), nutrients, gene expression (e.g., if one or more genes is specifically expressed/induced/repressed in one of the samples)), etc.
  • drugs/compounds e.g., growth factors, hormones, small chemical molecules
  • temperature change e.g., temperature change, osmotic shock, pH change, light
  • nutrients e.g., if one or more genes is specifically expressed/induced/repressed in one of the samples
  • the disclosure also provides a method for comparing the post- translational modification state of a lysine of a polypeptide between a first and second sample, preferably wherein said first sample is subjected to a physical, mechanical, or chemical treatment (e.g., treatment to drugs/compounds (e.g., growth factors, hormones, small chemical molecules), temperature change, osmotic shock, pH change, light etc.), the method comprising:
  • the first sample may be obtained from a patient having a specific condition or disorder, or from an animal or cell culture model system for said specific condition or disorder, whereas the second sample may be obtained from a healthy or wild-type sample. Disorders associated with altered lysine PTM are slowly being discovered.
  • the tumor suppressor gene VHL (von Hippel-Lindau) acts as a substrate receptor for a CUL2-based ubiquitin ligase and mutations in this gene are associated with lung cancer, sporadic clear cell renal carcinomas and an autosomal dominant familial cancer known as von Hippel-Lindau disease.
  • said specific condition or disorder is selected from cancer, an immune disorder, a
  • neurodegenerative disorder a metabolic disorder, a muscle wasting disorder, inflammation, or cardiovascular disease.
  • a first and second sample may be obtained from a patient having a specific condition or disorder, or from an animal or cell culture model system for said specific condition or disorder.
  • the first sample is treated with a test compound followed by steps a) through d).
  • a change in protease cleavage of a polypeptide between the first and second sample indicates that said treatment has an effect on the PTM state of a lysine.
  • the above method is also useful for screening for compounds that modulate the post- translational modification state of a lysine of a polypeptide.
  • a method for determining whether a compound modulates the post- translational lysine modification state of one or more polypeptides comprising contacting a first sample with a compound, and further comprising the steps:
  • the methods are useful for identifying compounds that modulate the post- translational lysine modification state of one or more polypeptides, particular the modification state of a particular polypeptide.
  • Such compounds may be useful as therapeutic agents, for example, if the presence or absence of a lysine PTM is the cause of a disorder then a compound that restores the lysine PTM to its "wild-type state" would be a useful treatment.
  • such methods are also useful for identifying compounds that do not modulate the post-translational lysine modification state of one or more polypeptides, particular the modification state of a particular polypeptide.
  • a drug discovery project one object is to identify compounds which are specific and have limited side- effects. Screening a candidate drug for its inability to affect the lysine PTM state of polypeptides reduces the chance of unforeseen side effects.
  • Suitable compounds for use in the methods include, growth factors, hormones, small chemical molecules, polypeptides, peptides, antibodies, nucleic acids (e.g., genes, cDNA's, RNA's, antisense molecules, siRNA/miRNA constructs, ribozymes, etc.), toxins, and combinations thereof.
  • the methods comprise a further step f) in which one or more peptides are identified which are present in only one of the samples.
  • the post-translational modifications which can be detected are those which render the peptide bond between the lysines and an adjacent amino acid resistant to proteolytic cleavage.
  • the PTM of lysine is selected from alkylation (e.g., methylation), acetylation, propionylation, butyrylation, ADP- ribosylation, ubiquitination, SUMOylation or modification by other ubiquitin-like proteins, including Nedd8, Fubi, Hub l, ISG15, FAT 10, URM1, UFM1, SAMP 1, SAMP2 or PUP.
  • the PTM of lysine is selected from alkylation (preferably methylation), acetylation, propionylation, ADP-ribosylation, ubiquitination,
  • ubiquitin-like proteins selected from Nedd8, ISG15, and UFM1. More preferably, the specific PTM of lysine is
  • Sumoylation refers to the addition of a SUMO protein (small ubiquitin like modifier) to a polypeptide.
  • Ubiquitination refers to the addition of ubiquitin to a polypeptide.
  • Lysine acetylation refers to the acetylation of lysine residues.
  • Alkylation refers to the addition of an alkyl group, preferably a methyl group, to a lysine residue. Acetylation is described in Choudhary et al. Science, 2009, 325:834-840. Propionylation, and butyrylation are described, e.g., in Chen et al., Molecular & Cellular Proteomics, 2007, 6:812-819.
  • the sample may comprise a single polypeptide or a collection of different polypeptides, such as the entire proteome.
  • Samples may be environmental, industrial, veterinary or medical in origin and from an animal, plant, a bacterium, a fungus, a protist or a virus.
  • the preferred samples include saliva, mucous, tears, blood, serum,
  • lymph/interstitial fluids are mammalian, more preferably human, serum and urine.
  • the sample is a cell culture in which the cells have been genetically engineered, e.g., to express a tagged SUMO protein (e.g., SUM02) or a tagged ubiquitination protein.
  • the methods comprise a step of chemically modifying essentially all lysines having a free epsilon amino group in said sample.
  • the PTMs of lysines described herein do not have free epsilon amino groups.
  • the chemical modification renders the peptide bond between said lysines and an adjacent amino acid resistant to proteolytic cleavage.
  • said peptide bond is resistant to cleavage to trypsin, Lys-C, and/or Lys-N.
  • Suitable chemical modifications include acetylation, treatment with N-succinimidyl-N-methylcarbamate (SMMC), or a sulfo- N- hydroxysuccinimide- linked compound (sulfo-NHS).
  • SMMC N-succinimidyl-N-methylcarbamate
  • sulfo-NHS sulfo-NHS-biotin.
  • Another way to chemically modify proteins and peptides uses citraconic anhydride. This results in a carboxylation of all free lysines, and this modification is reversible by low pH.
  • the chemical modification includes dimethylation, e.g., from the treatment of formaldehyde in the presence of NaBH3CN (see, e.g., Kleifeld et al.
  • a further chemical modification is carbamylation.
  • carbamylation For example, a sample is treated with a solution of 8 M urea at a pH of 8.5 and incubating for 4 h at 80 degrees C. Under these conditions, carbamylation occurs only on the primary amines of the peptides (see, e.g., Angel and Orlando, Rapid Commun Mass Spectrom. 2007;21(10): 1623-34).
  • a preferred chemical modification is acetylation of said lysine residues.
  • Lysine residues can be acetylated using, for example, sulfosuccinimidyl acetate, as described in the examples.
  • the chemical modification is acetylation and the PTM is selected from alkylation (e.g., methylation), propionylation, butyrylation, ADP- ribosylation.
  • alkylation e.g., methylation
  • propionylation e.g., butyrylation
  • ADP- ribosylation ubiquitination, SUMOylation or modification by other ubiquitin-like proteins, including Nedd8, Fubi, Hubl, ISG15, FAT 10, URM1, UFM1.
  • the methods described herein further comprise enriching for polypeptides comprising said specific PTM.
  • This step may be performed before, after, or during the step of chemically modifying step described above and before removing a single type of PTM from said post-translationally modified lysine.
  • said step is performed before the chemical modification step.
  • enriching refers to increasing the number of polypeptides comprising said specific PTM as compared to the number of polypeptides not comprising said specific PTM. This enrichment step provides a smaller and more convenient sample size for chemical modification purposes.
  • the enrichment step comprises contacting said sample with a binding molecule specific for said PTM.
  • said binding molecule is an antibody which recognizes said PTM.
  • the term "antibody” includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Methods of making antibodies are well known in the art and many suitable antibodies are commercially available. Exemplary antibodies include those directed to SUMO available from Santa Cruz Biotechnology, Inc.
  • SUMO-1 C- 19
  • SUMO-2 C-13
  • SUMO-3 M-20
  • Invitrogen SUMO- 1, 21C7
  • Abeam SUMO-2/3, 8A2
  • FK2 FK2, available from Millipore.
  • Antibodies to acetylated lysines are available from, e.g., Cell Signaling Technology (Acetylated- Lysine Antibody #9441) and antibodies to methylated lysines are available from, e.g., Abeam (Anti-Methylated Lysine antibody ab76118).
  • the PTM has been labeled, e.g., prior to lysine modification.
  • cells can be transfected with a SUMO or ubiquitin protein comprising a tag (e.g., Hisl0-SUMO2).
  • tags are known to one of skill in the art and include His, Flag, V5, myc, HA, GST, etc.
  • the binding body may also be specific for said tag, for example an anti-myc antibody in the case of a myc tag or nickel in the case of a His tag.
  • said enrichment step comprises contacting said sample with a binding molecule specific for said PTM, wherein said binding molecule is linked (directly or indirectly)to a solid support (e.g., beads).
  • a solid support e.g., beads
  • Targets of interest will thus be immobilized on the solid support.
  • the chemical modification step can be performed while said targets of interest are still immobilized on the solid support. After chemical modification, the solid support can simply be washed to remove any remaining chemical after the first modification step, and desalting or dialysis will not be required.
  • the methods described herein further comprise the step of removing a specific post- translational modification (PTM) from said post-translationally modified lysine, thereby rendering the peptide bond between said lysine and an adjacent amino acid susceptible to proteolytic cleavage.
  • PTM post- translational modification
  • This step results in a "freed” lysine.
  • a "freed lysine” refers to a lysine residue that initially contained a PTM which is subsequently removed during the detection method. Said "freed” lysine is susceptible to cleavage by proteases, such as trypsin, Lys-C, and Lys-N. Methods for removing a specific PTM are well-known to a skilled person.
  • said specific PTM is SUMOylation, and said PTM is removed by a SUMO- cleaving protease.
  • proteases include members of the Ulp enzyme family, e.g., Ulp l, Ulp2 (Smt4), SENP1, SENP2 (Axam, SuPr- 1, SMT3IP2), SENP3 (SMT3IP1), SENP5, SENP6 (SUSP1) or SENP7.
  • said SUMO-cleaving protease is SENP2.
  • said specific PTM is ubiquitination, and said PTM is removed by a deubiquitination enzyme (DUB) such as ubiquitin C-terminal hydrolases (UCHs) and ubiquitin-specific proteases (USPs/UBPs).
  • UCHs include BAP1 UCH-L1, Isopeptidase T/USP5, and UCH-L3.
  • Exemplary UBPs include UBP43/USP18, USP9x, USP2, USP14, USP7, USP25, and USP8. See also, Vertegaal 2011 Chem Rev 111 7923-7940 for exemplary JAB 1/MPN/MOV34 proteases (JAMMs) to remove ubiquitination.
  • JAB 1/MPN/MOV34 proteases exemplary JAB 1/MPN/MOV34 proteases
  • said specific PTM is alkylation which is removed by, e.g., a demethylase, in particular a member of the KDM family such as KDM1A, KDM1B, KDM2A, KDM2B KDM3A, KDM3B, JMJD IC, KDM4A, KDM4B, KDM4C, KDM4D KDM5A, KDM5B, KDM5C, KDM5D, KDM6A, KDM6B, and UTY.
  • a demethylase in particular a member of the KDM family such as KDM1A, KDM1B, KDM2A, KDM2B KDM3A, KDM3B, JMJD IC, KDM4A, KDM4B, KDM4C, KDM4D KDM5A, KDM5B, KDM5C, KDM5D, KDM6A, KDM6B, and UTY.
  • said specific PTM is acetylation which is removed by, e.g., a deacetylase (also referred to as lysine or histone deacetylases).
  • a deacetylase also referred to as lysine or histone deacetylases.
  • exemplary deacetylases include
  • step a the chemical modification (step a) should not be the same as the PTM. Therefore, when the specific PTM is acetylation, the chemical modification is not acetylation.
  • said specific PTM is ADP-ribosylation which is removed by, e.g.,
  • PARC Poly(ADP-ribose) glycohydrolase
  • said specific PTM is propionylation which is removed by, e.g., NAD-dependent protein deacetylase, has depropionylation activity in vitro.
  • said ubiquitin like PTM is Nedd8 which is removed by, e.g., NEDP1, COP9 signalosome, OTUB1, USP21, or BPLF1.
  • said ubiquitin like PTM is ISG15 which is removed by, e.g., USP18
  • said ubiquitin like PTM is UFM1 which is removed by, e.g., UfSPl and UfSP2.
  • the methods further comprise enriching for polypeptides in which a single type of post-translational modification (PTM) from said post- translationally modified lysine is removed.
  • PTM post-translational modification
  • This step occurs after the steps of a) chemically modifying essentially all lysines having a free epsilon amino group in said sample and b) removing a single type of PTM and enriches for polypeptides comprising lysines having a free epsilon amino group.
  • the peptide bond between the lysine (which previously was post-translationally modified) and an adjacent amino acid should still be susceptible to proteolytic cleavage after completion of this enrichment step.
  • the methods further comprise a step of proteolytically cleaving said polypeptide with a protease that cleaves a peptide bond between a lysine with a free amine group and an adjacent amino acid to produce a mixture of peptides.
  • a protease that cleaves a peptide bond between a lysine with a free amine group and an adjacent amino acid to produce a mixture of peptides.
  • said protease is trypsin which cleaves at the carboxyl side of arginine or lysine residues. A lysine residue which was "freed” from its specific
  • PTM is susceptible to trypsin digestion. Cleavage results in a peptide having a lysine or arginine residue at the C-terminal position and a peptide having as the N-terminal residue the amino acid which was previously on the carboxyl side of said lysine or arginine. It is this lysine residue which was the site of the specific post-translational modification.
  • said protease is Lys-C which cleaves at the carboxyl side of lysine residues.
  • a lysine residue which was "freed” from its specific PTM is
  • said protease is Lys-N which cleaves at the amino terminal side of lysine residues.
  • a lysine residue which was "freed” from its specific PTM is susceptible to Lys-N digestion. Cleavage results in a peptide having a lysine at the N-terminal position and a peptide having as the C-terminal residue the amino acid which was previously on the amino terminal side of said lysine. It is this lysine residue which was the site of the specific post-translational modification. It is clear to a skilled person that lysine residues which occupy the extreme N-terminus of a particular polypeptide will always be found in a N-terminal position of a peptide after cleavage with Lys-N.
  • an additional protease may also be used in order to generate small peptide fragments.
  • an additional protease may also be used in order to generate small peptide fragments.
  • smaller peptide fragments are preferred.
  • said peptides are between 5 and 100, 5 to 50, or 5 to 20 amino acids.
  • the detection method is top down mass spectrometry which does not require the use of small peptide fragments (see, e.g., Cui et al. Analyst, 2011, 136, 3854-3864).
  • the methods described herein further comprise the step of detecting the peptides produced from the proteolytic cleavage step. Detection of the peptides includes identifying the amino acid sequence of the peptides, determining the (apparent) molecular mass of the peptides, as well as determining the fragmentation pattern of the peptide fragments.
  • the peptide detection step comprises determining the fragmentation pattern of the peptide fragments.
  • the fragmentation pattern can be determined, e.g., by mass spectrometry or gel electrophoresis (e.g. two-dimensional electrophoresis), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), capillary gel electrophoresis, affinity chromatography, and Edman degradation. Determining the fragmentation pattern is especially useful when comparing the post-translational modification state of lysines between two different samples. The fragmentation pattern may also be compared to a known or predicted fragmentation pattern.
  • the peptide detection step comprises determining at least part of the amino acid sequence of one or more of the peptide fragments. Sequencing can be accomplished using any suitable technique, such as Edman degradation or mass spectrometry.
  • mass spectrometry methods are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.
  • Mass spectrometry methods are known to one in the art and are described in the examples herein, U.S. Patent Publication 20130122516, U.S. Pat. No. 5,719,060.
  • the peptides may be ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios.
  • Detection of a peptide typically will involve detection of signal intensity. Thus, both the quantity and mass of the peptide can be determined.
  • Data generated by desorption and detection of peptide can be analyzed with the use of a programmable digital computer.
  • the computer program analyzes the data to indicate the number of peptide detected, and optionally the strength of the signal and the determined molecular mass for each peptide detected.
  • peptide mass fingerprinting is used.
  • the mass of each peptide is determined, for example with MALDI-TOF, and then the masses are compared to a database containing theoretical or previously determined peptide masses. For example, Andromeda (Cox et al.
  • J Proteome Res., 2011, 10: 1794- 1805) and MaxQuant can be configured to generate and search against a peptide database in which every single lysine is acetylated. Further, the software may then be configured to allow a variable de-acetylation on any lysines which are at the C-terminus of a peptide. This allows a database to be generated which realistically matches up to the experimentally generated and measured set of peptides.
  • the peptide detection step comprises determining the molecular mass of the peptides, e.g., by gel electrophoresis or Western blot analysis.
  • antibodies to proteins known or suspected of having PTM can be used to detect a specific peptide fragment on a Western blot.
  • the methods described herein further comprising SILAC (stable isotope labeling with amino acids in cell culture) labeling of lysine residues.
  • SILAC stable isotope labeling with amino acids in cell culture
  • two populations of cells are grown in cell culture, each being supplemented with a different set of amino acids.
  • one population of cells may be grown with normal amino acids while the other population is supplemented with growth medium labeled with stable (non-radioactive) isotopes.
  • each population is supplemented with amino acids comprising a different isotope of lysine.
  • An exemplary SILAC labeling procedure is as follows. One set of cells is grown in normal media with natural amino acids ("light label", this group is optional); a second set of cells is grown with 2H4-lysine and 13C6- arginine ("intermediate label"/K4R6); and a third set of cells is grown in 15N2 13C6- lysine and 15N413C6-arginine ("heavy label"/K8/R10).
  • differentially labeled amino acids results in the production of a mass difference between proteins, and after proteolytic digestion, a mass difference between corresponding peptide pairs. These differences can be detected using mass spectrometry.
  • one population of cells is labeled with the intermediate label and another population of cells is labeled with the heavy label. This step is
  • the methods as described herein are performed on cell lysates, including mass spectrometry.
  • the peptide peaks of the differentially labeled peptide pairs can be very accurately quantified relative to each other to determine the peptide ratios.
  • Peptides having a ratio of 1 (or nearly 1) indicate "positive hits". Further details of SILAC labeling are given in the examples. Peptides having a ratio which differs substantially from 1 are likely not relevant as they may be due to incomplete acetylation or a computational error from the searching against a large database of peptides. All identified peptide pairs with a free C-terminal lysine, and peptide pairs which would have been preceded by a lysine on their N-terminal side, are indicative of lysines which were previously modified by the PTM.
  • the invention further provides a method for detecting whether a protein contains a post-translational modification said method comprising
  • binding molecules binds said protein depending on the presence or absence of the post- translation modification and the other binding molecule binds said protein
  • the post-translational modification can be a protein phosphorylation or other post- translational modification.
  • the post-translational modification is a proteinaceous post-translational modification.
  • the denaturing step is typically done to inactivate enzymes that can affect post-translational modification such as for instance a deconjugating enzyme or a phosphatase.
  • a deconjugating enzyme is a SUMO -protease.
  • the sample can be any sample that comprises a protein. It is preferred that the sample comprises different proteins.
  • the sample preferably comprises a complex mixture of proteins. Considering that the method comprises binding of said protein depending on the presence or absence of the post-translation modification it is preferred that the sample is, or is derived from a cell sample. Such samples contain proteins that have been produced in a cell, which typically is able to provide the protein with a post-translational modification.
  • the sample is preferably a tissue sample. Tissues in this context are tissues normally present in a healthy human or animal. Typical examples are skin, intestine, blood or cell fraction thereof, prostate, liver and the like. The list is not intended to be limitative.
  • Tissue in this context also includes cancer tissue or other cell sample not normally present in a healthy human or animal, such as a neoplasia or virus-induced growth such as a papilloma or wart.
  • the sample can also be a sample of a body fluid comprising protein.
  • Non-limitative examples are serum, plasma and urine.
  • the surfactant is preferably an anionic surfactant, preferably an organosulfate.
  • the organosulfate is preferably an
  • Organosulfates are a class of organic compounds that share the structure R-0-S(03) " .
  • the R-group is preferably an alkyl.
  • the alkyl is preferably a Cs-Ci6 alkyl, preferably a C9-C15 alkyl, more preferably a C10-C14 alkyl, more preferably a C11-C13 alkyl.
  • the alkyl is a C12 alkyl.
  • the alkyl is preferably a saturated linear alkyl.
  • the surfactant is sodium dodecyl sulphate, or an equivalent thereof. Suitable equivalents are at least different salts thereof.
  • the ammonium salt is also an equivalent.
  • the sample when herein below reference is made to a sample when referring to amounts or ratio's the sample is the original sample provided in step i, the sample comprising the surfactant of step ii, and the sample of step iii comprising the surfactant and the capture protein.
  • the surfactant added in step ii is preferably added to the original sample of step i, in an amount effective in denaturing protein in the sample.
  • the surfactant is added in excess of 0.5% weight/weight (surfactant versus the total weight of the sample comprising the surfactant).
  • the surfactant is added in amount of 1% weight/weight (of the total weight of the sample comprising the surfactant).
  • the surfactant is preferably added in an amount that does not exceed 10% and preferably not exceed the 2% weight/weight (of the total weight of the sample comprising the surfactant).
  • the capture protein captures surfactant that is not bound to protein and reduces the amount of free surfactant (i.e. not bound to protein), to an amount or concentration that does not interfere with the binding of a binding molecule to the protein of interest.
  • the capture protein is preferably added in an amount sufficient to reduce the amount of free surfactant to 0.1% or less (of the total weight of the sample comprising the surfactant and the capture protein). For instance, solutions comprising 0.1 %SDS typically do not interfere with the binding of a typical antibody to a protein of interest.
  • binding molecules are defined as defined herein above.
  • Preferred binding molecules are so-called “traps” or "TUBEs”.
  • traps or TUBEs are proteins that bind for instance SUMOylated or ubiquinated proteins via the post-translational modification.
  • Such binding molecules are presently also used in detection assay's (see for instance: SUMO-traps: referentie: Da Silva-Ferrada E et al. Sci Rep 2013, 3: 1690 doi: 10.1038/srep01690) or (TUBEs: Tandem Ubiquitin Binding Entities; referentie Lopitz-Otsoa F et al. 2012 J Proteomics.75:2998-3014. doi:
  • One of the first or second binding molecule binds said protein depending on the presence or absence of the post-translation modification and the other binding molecule binds said protein irrespective of the presence or absence of said post translational modification.
  • binding molecules that bind a protein depending on whether the protein comprises a post-translational modification at the binding site of the binding molecule.
  • binding molecules and antibodies thus bind to the protein only when it comprises the post-translational modification or only when it does not comprise the post-translational modification.
  • binding molecules are herein referred to as binding molecules that bind the protein depending on the presence or absence of the post-translation modification.
  • the binding molecule that that binds the protein depending on the presence or absence of the post-translation modification is a binding molecule that binds to the protein only when it contains a proteinaceous post-translational modification.
  • a binding molecule is preferably a binding molecule specific for the proteinaceous post-translation modification.
  • a binding molecule is preferably a binding molecule that binds to an antigen on the proteinaceous post-translation modification, optionally together with the linking amino acid on the main polypeptide chain of the protein.
  • Binding molecules preferably antibodies, that bind irrespective of the presence or absence of the post- translational modification typically bind to a site on the protein that is not a site of post-translational modification and/or can bind irrespective of the presence of the post-translational modification at the site.
  • modification is preferably a binding molecule that binds to a site on the protein that is not a site of post-translational modification. It is preferred that said first binding molecule binds said protein depending on the presence or absence of the post- translation modification and the said second binding molecule binds said protein irrespective of the presence or absence of said post translational modification.
  • the invention further provides a method for detecting whether a protein contains a post-translational modification said method comprising
  • step iii contacting the sample of step iii with said first antibody; removing unbound protein from the sample;
  • one of the first or second binding molecule binds an antigen on the proteinaceous post-translation modification and the other binding molecule binds an antigen on said protein.
  • Both binding molecules may bind an antigen (albeit a different antigen) on a proteinaceous post-translational modification. It is preferred, however, that one of the binding molecules binds an antigen on the protein different from an antigen on said proteinaceous post-translational modification. In this way one binding molecule binds an antigen on said proteinaceous post-translational modification and the other binding molecule binds an antigen on the protein different from an antigen on said proteinaceous post-translational modification.
  • the binding molecule that binds an antigen on the protein different from an antigen on said proteinaceous post-translational modification can be an antigen that comprises a post- translational modification or an antigen that has been produced by translation of the protein only.
  • the binding molecule binds an antigen that comprises a post- translational modification it does not bind an antigen on same the proteinaceous molecule as the other binding molecule.
  • the other binding molecule binds an antigen on the protein that is conjugated to the SUMO protein. This latter binding molecule for instance binds an antigen on the protein that is produced by translation.
  • the antigen that is not present in the proteinaceous translational modification is an antigen that has been produced by translation.
  • Antigens on the protein that do not comprise a post-translational modification are also referred to as translated antigens.
  • a binding molecule that binds an antigen on a proteinaceous translational modification is preferably completely present in said proteinaceous post-translational modification. In other words, the binding molecule preferably does not, for binding, require binding to amino acid residues that are not present in the proteinaceous post-translational modification.
  • the binding molecules are preferably antibodies or derivatives thereof.
  • the invention provides a method for detecting whether a protein contains a, preferably proteinaceous, post-translational modification said method comprising
  • the first binding molecule (or first antibody) preferably binds an antigen on a proteinaceous post-translation modification or preferably binds said protein depending on the presence of the post-translation modification.
  • all proteins comprising the post-translational modification with the antigen are bound by the first binding molecule (or first antibody). All other proteins and optionally unbound first binding molecules are removed from the sample.
  • the second binding molecule or antibody binds an antigen that is not present in the proteinaceous translational modification that the first binding molecule (or antibody) binds to.
  • the second binding molecule (or second antibody) binds an antigen on the proteinaceous post-translation modification.
  • the first binding molecule preferably binds an antigen on the protein that is not present in the proteinaceous post-translational modification that the first binding molecule binds to.
  • the second binding molecule can then be used to detect the proteinaceous post- translational modification. This setting can, for instance, be used to determine the amount of protein that comprises the post-translational modification.
  • the first antibody is preferably linked to a solid surface.
  • the linkage is preferably a physical linkage.
  • the immobilization to a solid surface can be used to easily separate bound from unbound protein.
  • the immobilization also facilitates washing steps.
  • the solid surface can be any solid surface that is compatible with binding molecule binding studies. Such solid surfaces are known to the skilled person and include, but are not limited to beads, various plastics and glass.
  • the solid surface is the protein binding surface of an ELISA plate.
  • a typical ELISA plate is a polystyrene microtiter plate.
  • the solid surface is a bead, preferably a magnetic bead or fluorescent bead.
  • the proteinaceous post-translational modification can be any proteinaceous modification that is physically linked to the protein of interest. Post-translational modifications share the common property that they can be used to modify a variety of different proteins. Ubiquitin is the most-understood proteinaceous post- translation modifier. There is, however, a growing family of ubiquitin- like proteins (UBLs) that modify cellular proteins in ways similar to, but distinct from, that of ubiquitin.
  • UBLs ubiquitin- like proteins
  • UBLs include: small ubiquitin-like modifiers (SUMOs), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene- 15 ISG15), ubiquitin- related modifier- 1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub l in S. cerevisiae), human leukocyte antigen F-associated (FAT 10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin- like protein (FUB1), MUB (membrane -anchored UBL) ubiquitin fold-modifier- 1
  • a post-translational modification is said to be proteinaceous when the unit attached to the protein comprises a peptide chain of at least 10 consecutive amino acids. It is preferred that the binding molecule that binds an antigen on a proteinaceous post-translational modification binds a ubiquitin-like protein.
  • the ubiquitin-like protein is preferably a SUMO protein or a ubiquitin protein.
  • the antigen of the proteinaceous post-translational modification is preferably an antigen as it is present when associated to the protein.
  • the association to the protein can be direct, or via another proteinaceous post-translational modification.
  • a ubiquitination can comprise a chain of individual ubiquitins linked to each other.
  • the antigen that is present on the post-translational modification can be present on a tag that is present on the proteinaceous post-translational modification.
  • cells can be provided with a tagged SUMO protein as indicated in the examples.
  • the antigen bound by the binding molecule can be an antigen present on the tag or the SUMO protein itself.
  • said proteinaceous post- translational modification comprises a tagged SUMO protein or tagged ubiquitin protein.
  • the binding molecule that binds an antigen on the proteinaceous post-translational modification binds an antigen on the tag.
  • the tag is preferably an epitope tag, a short peptide sequence which is selected because high-affinity antibodies can be reliably produced.
  • Epitope tags can be derived from viral genes, which explains their high immunore activity. Epitope tags include but are not-limited to a V5-tag, a Myc-tag, a HA-tag or a FLAG tag.
  • the capture protein can be any protein. It typically does not comprise the post- translational modification that the binding molecule is specific for.
  • the capture protein is preferably a protein that can be produced with a high standard quality.
  • the capture protein comprises a serum albumin, preferably bovine or human serum albumin.
  • the capture protein is preferably serum albumin, preferably bovine serum albumin.
  • the capture protein is preferably added in an amount sufficient to allow binding of the first binding molecule to the protein.
  • the actual amount can vary. For instance, depending on the amount of surfactant used, the amount of protein in the sample at the start of the procedure, the type of binding molecule used, and the particular antigen bound. Suitable amounts that apply to a large number of different
  • the sample when contacted with the first binding molecule, contains 0.1- 10% total protein and 0.2-4% surfactant, preferably 0.1-5% total protein and 0.2-4% surfactant, preferably 0.2-4% total protein and 0.3-3% surfactant, preferably 1-3% total protein and 0.4-2% surfactant.
  • the ratio of total protein to surfactant present in the sample of step iii is preferably from 1: 1-10: 1; preferably 2: 1- 5: 1 and more preferably 5: 1 (total protein/surfactant in weight/weight)
  • the % total protein and surfactant are given in weight in relation to the weight of total sample prepared in step iii.
  • Total protein comprises the amount of capture protein used and the amount of protein of the sample (sample protein) as it is provided to be used in a method for detecting whether a protein contains a
  • sample protein post-translational modification of the invention.
  • the ratio of sample protein versus capture protein is preferably from 10: 1 to 1: 10, preferably 8: 1 to 1:5, preferably 4: 1 to 1:2 sample apture protein.
  • the capture protein is preferably added in amount of at least 20% and preferably at least 40%, more preferably at least 60%, more preferably at least 100% relative to the amount of surfactant present in the sample of step iii (% given in weight/weight capture protein/surfactant).
  • a cell sample is suspended in 4 volumes of a solution comprising 1% surfactant.
  • This sample typically contains about 4% protein.
  • the sample comprising denatured protein is subsequently diluted with an equal volume comprising 1% capture protein.
  • a sample as used herein may further contain various additives such as salt, buffer, protease inhibitors and the like.
  • a method for detecting whether a protein contains a post-translational modification of the invention can be used for a variety of applications.
  • a method for detecting whether a protein contains a post-translational modification of the invention is used in determining whether the protein is derived from a cell comprising damaged DNA.
  • DNA damage has been shown to alter the extend of modification of proteins by post-translational modifications.
  • a method for detecting whether a protein contains a post-translational modification of the invention can thus be used to test samples for the presence of cells that have suffered DNA damage.
  • a method of the present invention can thus be used to screen compound libraries for the presence of compounds that affect the extend of modification of proteins by post-translational modifications.
  • the invention therefore provides a method for determining whether a compound affects the post-translation modification and/or the extend of post- translational modification of a protein by a post-translational modification, said method characterized in that said protein is detected by a method for detecting whether a protein contains a proteinaceous post-translational modification of the invention.
  • the methods can be used to screen compound libraries for compounds that inhibit or activate conjugating or deconjugating enzymes.
  • the methods can also be used for biosafety testing.
  • a method of the invention is quick and can indicate early on whether the compound or composition is likely to be safe.
  • a method of the invention is preferably used in a high-throughput assay. Compounds and compositions that are not safe, or likely not safe can be tracked down and identified at an early stage of development. Further definitions
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • an element means one element or more than one element.
  • Example 1 Lysine protection assay (acid elution, biotinylation, western blot analysis)
  • HeLa cell line stably expressing HislO-SUM02 (A11KR-Q87R mutant) was generated. HeLa cells were grown in a 6-well plate containing Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • cells were sorted according to their GFP fluorescence using a BD FACSAria II, at a rate of -5.000 cells per second, into two fractions with a low GFP intensity (GFP BF 530/30-A intensity 10 ⁇ 3.0-10 ⁇ 3.3) and a high GFP intensity (GFP BF 530/30-A intensity 10 ⁇ 3.9- 10 ⁇ 4.2).
  • Fluorescence-sorted cells were cultured in a T25 flask until confluent, subsequently passed to a T75, and finally to a T175 flask. After reaching full confluence in the T175 flask, cells were harvested and re-suspended in DMEM with 10% FBS, 10% DMSO and P/S and slowly frozen at -80 Celsius.
  • HeLa cells stably expressing HislO-SUM02 (at a low level) were seeded in 3x 15-cm dishes containing regular DMEM supplemented with 10% FBS and P/S, at a confluence of 10%. After 3 days of growth, cells were harvested and the pellet (0.3 mL) was vigorously lysed in 25 pellet volumes (7.5 mL) of 6M guanidine-HCl, 100 mM sodium phosphate, 10 mM TRIS, at pH 8.0. Immediately following lysis, the lysate was snap frozen and stored at -80 Celsius until further processing.
  • the lysate was thawed at 25 Celsius and sonicated at power 7.0 (-30 Watt) for 5x 5 seconds, mixing the sample in between sonication steps. Following sonication, the lysate was tumbled at room temperature for 30 minutes. Subsequently, beta- mercaptoethanol was added to a concentration of 5 mM, and imidazole (pH 8.0) was added to a concentration of 50 mM.
  • Ni-NTA beads (200 ⁇ L bead volume) were washed (4x) and equilibrated in 6M guanidine-HCl, 100 mM sodium phosphate, 10 mM TRIS, at pH 8.0, supplemented by
  • Ni-NTA beads 5 mM beta-mercaptoethanol and 50 mM imidazole pH 8.0. The equilibrated Ni-NTA beads were added to the lysate, and tumbled for 5 hours at room temperature.
  • the beads were pelleted by centrifugation at 500 RCF. Next, the beads were washed (re-suspended in a buffer, gently mixed for 10 seconds, and then re-pelleted at 500 RCF) in order with the following buffers:
  • SNHSA Sulfosuccinimidyl Acetate
  • the SNHSA was weighed and kept in separate tubes, and only added to the AB at the final moment, upon which it was swiftly mixed and instantly added to the Ni-NTA beads.
  • the beads were swiftly re-suspended and tumbled for 10 minutes, upon which 1 ⁇ of 6M NaOH was added to raise the pH back up to -8.0.
  • a second acetylation round was performed by adding another bead volume of AB supplemented with 20 mM fresh SNHSA to the mixture. Following 10 minutes of tumbling, 3 6M NaOH was added to raise the pH over 8.0 and finalize the reaction. Subsequently, the beads were tumbled for an additional 10 minutes.
  • TRIS pH 8.0 was added to a final concentration of 20 mM to quench any remaining SNHSA.
  • the beads were pelleted at 500G and subject to the following washes in order:
  • the proteins were eluted off the Ni-NTA beads using one buffer volume (200 ⁇ ) of 56 mM citric acid, 44 mM sodium citrate, 8M urea, at pH 4.4 (elution buffer). After addition of the elution buffer, the beads were tumbled for 15 minutes to ensure proper suspension of the beads and elution of the proteins. Next, the beads were pelleted at 500G and the eluted proteins were transferred to a separate tube. The beads were then re-suspended in one additional buffer volume of elution buffer, for an additional 15 minutes, to yield a second fraction of eluted proteins.
  • the sample was slowly diluted to 3M urea by addition of 4 volumes of 1.75M urea, 100 mM sodium phosphate, at pH 8.5, gently mixing the sample while diluting to prevent abrupt changes in buffer condition. Subsequently, 15 ⁇ g (1.500 U) of dialyzed (primary- amine depleted) recombinant HislO-SENP2-CD (catalytic domain) was added to the sample. The sample was gently mixed and left for 48 hours at room temperature, in the dark, and undisturbed.
  • Triton X- 100 was added to a concentration of 0.5%, and the sample was heated to 37 Celsius for 30 minutes to precipitate HislO-SENP2-CD. Following incubation, the sample was centrifuged at 10.000 RCF to clear precipitated SENP2. The sample was then transferred to a clean tube and treated with 1 mg (0.83 mM) of Sulfosuccinimidyl-SS-Biotin (SNHSSSB). SNHSSSB was immediately added to the sample after being dissolved in 8M urea, 100 mM sodium phosphate pH 8.5.
  • TRIS pH 8.0 was added to a final concentration of 50 mM, and the sample was incubated for another 30 minutes at 30 Celsius to quench any remaining SNHSSSB.
  • One volume of 8M urea was added to the sample to raise the overall concentration of urea to 5M.
  • neutravidin beads 200 ⁇ were washed (4x) and equilibrated in 8M urea, 100 mM sodium phosphate, 10 mM TRIS, 0.2% Triton X-100, at pH 8.5 (neutravidin wash buffer). The equilibrated neutravidin beads were added to the sample and tumbled for 3 hours at room temperature. Following incubation, the beads were pelleted by centrifugation at 500 RCF, and washed 6x with neutravidin wash buffer. Following washing, the neutravidin beads were eluted for 10 minutes at 30 Celsius with one bead volume of neutravidin wash buffer supplemented with 100 mM of dithiothreitol (DTT). A secondary elution was performed using one bead volume lx LDS Sample Buffer (NuPAGE) supplemented with 100 mM DTT, for 15 minutes at 50 Celsius.
  • DTT dithiothreitol
  • HeLa cell line stably expressing HislO-SUM02 (wild-type) was generated.
  • HeLa cells were grown in a 6-well plate containing Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • cells were infected using a lentiviral vector containing CMV-HislO- SUM02-IRES-GFP at a Multiplicity of Infection of 2, supplemented by 8 ⁇ g/mL of polybrene.
  • the medium was changed to standard DMEM with 10% FBS and P/S.
  • cells were trypsinized using Trypsin/EDTA and passed to a T75 flask. After again reaching full confluence, cells were similarly passed to a T175 flask
  • cells were sorted according to their GFP fluorescence using a BD FACSAria II, at a rate of -5.000 cells per second, into two fractions with a low GFP intensity (GFP BF 530/30-A intensity 10 ⁇ 3.0- 10 ⁇ 3.3) and a high GFP intensity (GFP BF 530/30-A intensity 10 ⁇ 3.9-10 ⁇ 4.2).
  • Fluorescence-sorted cells were cultured in a T25 flask until confluent, subsequently passed to a T75, and finally to a T175 flask. After reaching full confluence in the T175 flask, cells were harvested and re-suspended in DMEM with 10% FBS, 10% DMSO and P/S and slowly frozen at -80 Celsius.
  • HeLa cells stably expressing HislO-SUM02 (at a low level) were seeded in 4x 15-cm dishes containing either intermediate SILAC DMEM (K4R6) or 4x 15-cm dishes containing heavy SILAC DMEM (K8R10), at a confluence of 25%. After 3 days of growth, all cells were trypsinized, washed twice with PBS, and split to 20x 15-cm dishes containing either intermediate or heavy SILAC DMEM.
  • the lysates were thawed at 25 Celsius and sonicated at power 7.0 (-30 Watt) for 2x 5 seconds per 15 mL lysate, mixing the sample in between sonication steps. Following sonication, the lysates were tumbled at room temperature for 30 minutes. During this time, a bicinchoninic acid (BCA) assay was performed to measure the relative concentration of protein in the intermediate-labeled and heavy-labeled SILAC lysates. After quantification, lysates were pooled in an equimolar ratio.
  • BCA bicinchoninic acid
  • beta- mercaptoethanol was added to a concentration of 5 mM, and imidazole (pH 8.0) was added to a concentration of 50 mM. Finally, the lysate was divided over 12x 15 mL tubes containing 7 mL lysate each.
  • Ni-NTA beads (2 mL bead volume) were washed (4x) and equilibrated in 6M guanidine-HCl, 100 mM sodium phosphate, 10 mM TRIS, at pH 8.0, supplemented by 5 mM beta-mercaptoethanol and 50 mM imidazole pH 8.0. The equilibrated Ni-NTA beads were added to the lysate, and tumbled for 4 hours at room temperature.
  • the beads were pelleted by centrifugation at 500 RCF. The supernatant was removed, and all beads were pelleted in a single 15 mL tube. Next, the beads were washed (re-suspended in a buffer, gently mixed for 10 seconds, and then re-pelleted at 500 RCF) in order with the following buffers:
  • the beads were swiftly re-suspended and tumbled for 10 minutes, upon which 5 of 6M NaOH was added to raise the pH back up to -8.0.
  • a second acetylation round was performed by adding another bead volume of AB supplemented with 20 mM fresh SNHSA to the mixture.
  • 15 6M NaOH was added to raise the pH over 8.0 and finalize the reaction.
  • the beads were tumbled for an additional 10 minutes.
  • the beads were pelleted at 500G and subject to the following washes in order:
  • the proteins were eluted off the Ni-NTA beads using one buffer volume (2 mL) of 8M urea, 100 mM sodium phosphate, 10 mM TRIS and 500 mM imidazole, at pH 7.0. After addition of the elution buffer, the beads were tumbled for 30 minutes to ensure proper suspension of the beads and elution of the proteins. Next, the beads were pelleted at 500G and the eluted proteins were transferred to a separate tube. The beads were then re-suspended in one additional buffer volume of elution buffer, for an additional 30 minutes, to yield a second fraction of eluted proteins.
  • the purified acetylated HislO-SUM02 conjugated proteins were concentrated on a 100 kDa cut-off filter (twice washed with elution buffer). To facilitate concentration, only 400 of proteins were loaded on the filter per cycle, and then concentrated at 8.000 RCF for 10 minutes at a stabilized temperature of 20 Celsius. After each cycle, another 400 of proteins were loaded on top of the concentrate and concentrated in a similar fashion. After five cycles, concentration time for each cycle was increased to 20 minutes. After loading and concentration the final sample of proteins, an additional 20 minutes of concentration was performed in order to decrease the concentrate volume to approximately l/40 th of the starting volume (100 ⁇ ). The final concentrate was removed from the filter by placing it upside-down into a clean 1.5 mL tube and centrifuging it in a small table-top centrifuge at 500G for 30 seconds.
  • the sample was slowly diluted to 3M urea by addition of 4 volumes (400 ⁇ ) of 1.75M urea, 25 mM TRIS, at pH 7.0, gently mixing the sample while diluting to prevent abrupt changes in buffer condition. After dilution, DTT was added to a final concentration of 2 mM, and subsequently 25 ⁇ g (2.500 U) of recombinant HislO-SENP2-CD (catalytic domain) was added to the sample. The sample was gently mixed and left overnight at room temperature, in the dark, and undisturbed.
  • the concentration of urea was raised back up to 8M by addition of two volumes (1 mL) of 10.5M urea.
  • the sample was then concentrated over a 100 kDa cut-off filter (washed twice with 8M urea, 25 mM TRIS, at pH 7.0), as described previously, to approximately l/30 th of the starting volume (50 ⁇ ,) .
  • the final concentrated sample was digested according to the standard in-solution digestion protocol, briefly: Acetylated deSUMOylated proteins in 8 M urea were supplemented with ammonium bicarbonate (ABC) to 50 mM. Reduction and alkylation were performed with 1 mM dithiothreitol (DTT) and 5 mM chloroacetamide, for 30 minutes, respectively. Samples were then diluted 4-fold using 50 mM ABC. Subsequently, 1 ⁇ g of Sequencing Grade Modified Trypsin (Promega) was added to the samples. Digestion with trypsin was performed overnight, at room temperature, still and in the dark.. 25% of the digest was analyzed per run using nanoLC-MS/MS (Q-Exactive).
  • Samples were eluted off the RP column using a 4 hour gradient ranging from 0.1% FA to 80% ACN/0.1% FA. Positive scan mode was used, with default charge state 2. The resolution of full MS was 70,000, with an AGC target of 3e6 and a maximum IT of 20 ms. Scan range was 300 to 1750 m/z.
  • MS/MS the resolution was 17,500 with an AGC target of le5 and a maximum IT of 120 ms.
  • An underfill ratio of 0.1% was set, leading to a minimum intensity threshold of 8.3e2. Singly charged objects were rejected, and peptide matching was preferred.
  • a 45-second dynamic exclusion was used. Analysis of the raw data was performed using MaxQuant version 1.4.0.8..
  • HeLa cells stably expressing low levels of Flag-SUMO-2 were collected, spun down by centrifugation and washed with lx phosphate buffered saline (PBS) two times.
  • Cells were lysed in 4 pellet volumes of lysis buffer (1% SDS, 0.5% NP-40, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM ⁇ -glycerol phosphate, 5 mM sodium pyrophosphate, 0.5 mM EGTA, 5 mM 1, 10-phenanthroline in lx PBS (chemicals purchased from Sigma-Aldrich.
  • Protease inhibitors including EDTA were added (Roche; 1 tablet per 10 ml buffer). Lysates were frozen in liquid nitrogen and, if needed, stored at -80°C.
  • Lysates described above were thawed at 30°C and 70 mM chloroacetamide (Sigma-Aldrich) was added freshly. Samples were sonicated to reduce viscosity and incubated for 30 minutes at room temperature. An equal volume of dilution buffer (2% Triton X-100, 0.5% sodium deoxycholate, 1% BSA (bovine serum albumin), freshly added 70 mM chloroacetamide, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM ⁇ -glycerol phosphate, 5 mM sodium pyrophosphate, 0.5 mM EGTA, 5 mM 1, 10- phenanthroline, protease inhibitor+EDTA (Roche; 1 tablet per 10 ml buffer) in lx PBS) was added to the lysates and they were centrifuged for 15 minutes at 13200 rotations per minute at 4°C]. The supernatant was further used for the Sandwich Elisa.
  • Reagent diluent 0.4% bovine serum albumin in PBS
  • Substrate kit Color Reagent Pack R&D systems.
  • Small Ubiquitin-like Modifier is a post-translational modification
  • PTM lysine residues in proteins
  • Precursor SUMO is processed by SUMO-specific proteases in order to generate mature SUMO 7 , which is subsequently conjugated to target proteins through an enzymatic cascade involving the dimeric El activating enzyme SAEl/2, the E2 conjugation enzyme Ubc9, and several catalytic E3 enzymes 8 .
  • SAEl/2 dimeric El activating enzyme
  • Ubc9 the E2 conjugation enzyme
  • Ubc9 the E2 conjugation enzyme Ubc9
  • catalytic E3 enzymes 8 catalytic E3 enzymes 8 .
  • SUMOylation is often found to target lysines within the canonical consensus motif [VIL]KxE in proteins, and thus, unlike many other PTMs, shows a remarkable specificity in its conjugation behavior 9 10 .
  • SUMOylation of proteins is a reversible process, since SUMO-specific proteases can efficiently remove SUMO from its target proteins 1 .
  • SUMO is essential for the viability of all eukaryotic life, with the exception of some species of yeast and fungi 8 .
  • Ubc9 knockout mice perish at the early post- implantation stage due to chromosome condensation and segregation defects, further stressing the importance of SUMO in maintenance of the genome n .
  • SUMO-2 was found to be indispensable for the embryonic development of mice, whereas SUMO-1 and SUMO-3 knockout mice were still viable 12 .
  • SUMO-3 All SUMOs share the characteristic ubiquitin ⁇ -grasp fold, regardless of their limited sequence similarity to ubiquitin. Mature SUMO-2 and SUMO-3 are nearly identical 13 , differing by only three N-terminal amino acids, and are typically referred to as SUMO-2/3 as no antibody is able to distinguish between them. SUMO-1 only shares 47% sequence homology with SUMO-2/3, and is thus considered as a separate family member, although all SUMOs are conjugated to their targets by the same enzymatic machinery.
  • SUMO-2/3 are the more abundant forms of the SUMOs 14 , and a considerable amount of free SUMO-2/3 exists within the cell, allowing SUMO- 2/3 to function efficiently in response to cellular stresses or changes in growth conditions.
  • SUMO-1 is predominantly conjugated to RanGAPl.
  • SUMO-1 and SUMO- 2/3 share a significant overlap in conjugation targets, but also retain differential conjugation specificity 15 > 16 .
  • SIMs SUMO Interacting Motifs
  • STUbL Ubiquitin Ligase
  • Additional examples of SIM-mediated interactions include the interaction between SUMO-modified RanGAPl and the nucleoporin RanBP2 24 , and the localization of the transcriptional corepressor Daxx to PML nuclear bodies 25 .
  • SUMOylation has also become increasingly implicated as a viable target in a clinical setting s.26-29 j n a scre en for Myc-synthetic lethal genes, SAE1 and SAE2 were identified, indicative of Myc-driven tumors being reliant on SUMOylation 21 .
  • SAE1 and SAE2 were identified, indicative of Myc-driven tumors being reliant on SUMOylation 21 .
  • SAE1 and SAE2 were identified, indicative of Myc-driven tumors being reliant on SUMOylation 21 .
  • SUMOylation is widely involved in carcinogenesis 29 . Nevertheless, the system-wide knowledge of protein SUMOylation is limited to the global protein-level. Specific sites of SUMO modification have mainly been studied at the single protein level using low-throughput methodology.
  • proteomic approaches have elucidated hundreds of putative target proteins 15 > 19 > 30 > 31 , they have failed to elucidate SUMO acceptor lysines, with the notable exception of a recent study that revealed SUMOylation sites under heat stress conditions 32 .
  • PTMs 33 36 Various well-studied major PTMs include phosphorylation 37 > 38 , acetylation 39 , methylation 40 and ubiquitylation 41 45 , where tens of thousands of modification sites have been identified at the system-wide level.
  • site-specific identification of SUMOylation sites significantly trails behind these other PTMs, even though the amount of SUMO-modified proteins is predicted to be within the same order of magnitude as compared to other PTMs.
  • SUMO-2 mutants contain either the Q87R mutation, which is homologous to the sole yeast SUMO Smt3, or the T90R or T90K mutations, which are homologous to ubiquitin. Additionally, all internal lysines could be mutated to arginines to render the mutant SUMO immune to Lys-C. In turn, this allows for pre-digestion of the entire lysate and enrichment of SUMOylated peptides as compared to proteins, greatly diminishing the sample complexity. This approach has allowed for the identification of around 200
  • mutant SUMO Regardless of the success of approaches employing mutant SUMO, there are some drawbacks. Firstly, the substitution of all internal lysines to arginines prevents the mutant SUMO itself from being modified, effectively abrogating the ability to form polymeric chains. SUMOylation sites can be mapped while using just the Q87R or T90R mutation, but this hampers enrichment of modified peptides, a key step in the purification process for all other major PTMs. Secondly, the usage of mutant SUMO necessitates the usage of exogenous SUMO, and thus is incompatible with the identification of lysines modified by endogenous SUMO, for example from clinical samples or animal tissues.
  • PRISM involves chemical blocking of all free lysines in a complex sample, followed by treatment that removes a post-translational modification from post-translationally modified lysines thereby rendering the peptide bond between the lysine and an adjacent amino acid susceptible to proteolytic cleavage.
  • SUMOylation SUMOylated lysine are freed by SUMO-specific proteases.
  • the complex sample is treated with a proteolytic enzyme that cleaves a peptide bond between the "freed" lysines and an adjacent amino acid.
  • the HislO-SUMO-2 we described and used hereni has the following amino acid sequence:
  • DMEM Dulbecco's Modified Eagle's Medium
  • HeLa cell lines stably expressing wild-type His 10- SUMO-2 and lysine- deficient HislO-SUMO-2 (A11KR-Q87R mutant) were generated. To this end, HeLa cells were infected using a lentiviruses encoding CMV-[SUMO]- IRES-GFP. Following infection, cells were sorted for low GFP fluorescence using a FACSAria II (BD Biosciences).
  • FBS Fetal Bovine Serum
  • P/S penicillin and streptomycin
  • HeLa cells stably expressing HislO-SUMO-2 were seeded in 4x 15-cm dishes containing either medium SILAC DMEM
  • Lysates were sonicated at power 7.0 (-30 Watt) for 2x 5 seconds, using a microtip sonicator. Blocking of lysines with Sulfosuccinimidyl Acetate (SNHSA) and removal of SUMO using SUMO proteases in HeLa total lysate
  • HeLa cells were harvested and lysed in 8 M urea, 100 mM sodium phosphate, pH 8.5. Chloroacetamide was added to 10 mM. Blocking of all lysines was performed by addition of SNHSA to a concentration of 20 mM, for 30 minutes at room temperature. Afterwards, TRIS pH 8.0 was added to a concentration of 50 mM. Urea dilutions were performed using 100 mM sodium phosphate, pH 8.5. Removal of SUMO was performed using recombinant catalytic domains of SENP1 (LifeSensors) and SENP2 (LifeSensors). Digestion analysis of acetylated proteins was performed using
  • Lysates were supplemented with ⁇ -mercaptoethanol ( ⁇ - ⁇ ) to a concentration of 5 mM, and imidazole (pH 8.0) to a concentration of 50 mM.
  • 20 ⁇ Ni-NTA beads (Qiagen) were prepared per 1 mL of lysate, and subsequently washed (4x) and equilibrated in 6 M guanidine-HCl, 100 mM sodium phosphate, 10 mM TRIS, 5 mM ⁇ - ME, 50 mM imidazole, pH 8.0. Beads were added to lysates and tumbled for 5 hours at room temperature. Beads were washed with the following buffers. Wash Buffer 1 (2x): 6 M guanidine-HCl, 100 mM sodium phosphate, 10 mM TRIS, 10 mM imidazole,
  • AB Acetylation Buffer
  • proteins were eluted off the beads using one bead volume of 8 M urea, 56 mM citric acid, 44 mM sodium citrate, pH 4.4 (Elution Buffer). Beads were eluted twice for 15 minutes, and elutions were pooled, neutralized to pH 8.0 by addition of 6 M NaOH, and cleared by passage through 0.45 ⁇ filter columns (MilliPore). Imidazole elution of acetylated HislO-SUMO-2-conjugated proteins, for proteomics
  • proteins were eluted off the beads using one bead volume of 8 M urea, 100 mM sodium phosphate, 10 mM TRIS and 500 mM imidazole, pH 7.0. Beads were eluted twice for 15 minutes, and elutions were pooled and cleared by passage through 0.45 ⁇ filter columns (MilliPore).
  • Sample were gently diluted to 3 M urea by addition of 4 volumes of 1.75 M urea, 100 mM sodium phosphate, pH 8.5. After dilution, DTT was added to 2 mM.
  • samples were treated with 1 mg (0.83 mM) of Sulfosuccinimidyl-SS-Biotin (SNHSSSB). Following 2 hours of incubation at room temperature, TRIS pH 8.0 was added to a final concentration of 50 mM, and samples were incubated for another 30 minutes at 30 °C to quench any remaining SNHSSSB. Subsequently, the concentration of urea was increased to 5 M.
  • SNHSSSB Sulfosuccinimidyl-SS-Biotin
  • Neutravidin beads 200 of Neutravidin beads (Thermo) were washed (4x) and equilibrated in 8 M urea, 100 mM sodium phosphate, 10 mM TRIS, 0.2% Triton X- 100, pH 8.5 (Neutravidin Wash Buffer). The equilibrated neutravidin beads were added to the samples and tumbled for 3 hours at room temperature. Following incubation, the beads were washed 6x with Neutravidin Wash Buffer. Finally, beads were eluted for 10 minutes at 30 °C with one bead volume of Neutravidin Wash Buffer supplemented with 100 mM of dithiothreitol (DTT). A secondary elution was performed using one bead volume lx LDS Sample Buffer (NuPAGE) supplemented with 100 mM DTT, for 15 minutes at 50 °C.
  • DTT dithiothreitol
  • Protein samples were size-fractionated on Novex 4-12% Bis-Tris gradient gels using MOPS running buffer (Invitrogen), or on home-made 10% polyacrylamide gels using TRIS-Glycine buffer. Size-separated proteins were transferred to Hybond-C membranes (Amersham Biosciences) using a submarine system (Invitrogen). Gels were Coomassie stained according to manufacturer's instructions (Invitrogen).
  • Membranes were stained for total protein loading using 0.1% Ponceau-S in 5% acetic acid (Sigma). Membranes were blocked using PBS containing 0.1% Tween-20 (PBST) and 5% milk powder for one hour. Subsequently, membranes were incubated with primary antibodies as indicated, in blocking solution. Incubation with primary antibody was performed overnight at 4°C. Subsequently, membranes were washed three times with PBST and briefly blocked again with blocking solution. Next, membranes were incubated with secondary antibodies (donkey-anti-mouse-HRP or rabbit-anti-goat-HRP, Pierce) for one hour, before washing three times with PBST and two times with PBS. Membranes were then treated with ECL2 (Pierce) as per manufacturer's instructions, and chemiluminescence was captured using Biomax XAR film (Kodak).
  • Samples were analyzed by means of nanoscale LC-MS/MS using an EASY-nLC system (Proxeon) connected to a Q-Exactive (Thermo) using Higher- Collisional Dissociation (HCD) fragmentation. Samples were eluted off a reversed phase C18 column packed in-house, using a 4 hour gradient ranging from 0.1% formic acid to 80% acetonitrile/0.1% formic acid, at a flow rate of 250 nL per minute. The mass spectrometer was operated in data- dependent acquisition mode using a top 10 method. The resolution of full MS acquisition was 70,000, with an AGC target of 3e6 and a maximum injection time of 20 ms. Scan range was 300 to 1750 m/z.
  • MS/MS spectra were filtered and deisotoped, and the 15 most abundant fragments for each 100 m/z were retained. MS/MS spectra were filtered for a mass tolerance of 6 ppm for precursor masses, and a mass tolerance of 20 ppm was used for fragment ions.
  • Peptide and protein identification was performed through matching the identified MS/MS spectra versus a target/decoy version of the complete human Uniprot database, in addition to a database of commonly observed mass spectrometry contaminants. Up to 5 missed tryptic cleavages were allowed, to compensate for extensive internal acetylation within peptides due to the PRISM methodology.
  • Cysteine carbamidomethylation was set as a fixed peptide modification. Peptide pairs were searched with a multiplicity of 2, allowing medium labeled and heavy labeled SILAC peptides. Medium peptides were set to be labeled with Arginine-6
  • Heavy peptides were set to be labeled with Arginine-10 (monoisotopic mass of 10.008269) and Lysine-8-Acetyl (monoisotopic mass of 50.024763). Protein N- terminal acetylation and methionine oxidation were set as variable peptide modifications. Moreover, to allow identification of peptides ending in a "free” lysine, a "negative" weight acetyl (monoisotopic mass of -42.010565) was set as a variable peptide C-terminal lysine modification. Up to 5 peptide modifications were allowed.
  • Peptides were accepted with a minimum length of 6 amino acids, a maximum size of 4.6 kDa, and a maximum charge of 7.
  • the processed data was filtered by posterior error probability (PEP) to achieve a protein false discovery rate (FDR) of below 1% and a peptide-spectrum match FDR of below 1%.
  • Peptides ending with a lysine or being preceded by a lysine were assumed to be corresponding to previously
  • SUMOylated lysines Peptides were additionally filtered to have an Andromeda score of at least 40, and detected as either a medium or a heavy labeled SILAC peptide. Proteins for comparative analysis were only those containing at least one site. The putative list of SUMO target proteins is based on all peptides detected after HislO- SUMO-2 pulldown, and was filtered so proteins were identified by at least two peptides, one unique peptide, and adhered to an internal medium/heavy SILAC ratio in between 2/3 and 3/2. IceLogo and heatmap generation
  • IceLogo software version 1.2 56 was used to overlay sequence windows in order to generate a consensus sequence, which was compensated against expected occurrence (IceLogo). Heatmaps were generated in a similar fashion to IceLogos. All amino acids shown as enriched or depleted are significant with p ⁇ 0.05. Term enrichment analysis
  • Statistical enrichment analysis for protein and gene properties was performed using Perseus software 57 .
  • the human proteome was annotated with Gene Ontology terms 58 , including Biological Processes (GOBP), Molecular Functions (GOMF), and Cellular Compartments (GOCC). Additional annotation was performed with Keywords, GSEA, Pfam, KEGG and CORUM terms.
  • SUMOylated proteins were compared by annotation terms to the entire human proteome, using Fisher Exact Testing. Benjamini and Hochberg FDR was applied to p-values to correct for multiple hypotheses testing, and final corrected p-values were filtered to be less than 2%. Final scoring of terms was performed by multiplying the 2-Log of the enrichment ratio by the negative 10-Log of the FDR, which allowed ranking of terms by both their enrichment and confidence.
  • STRING network analysis was performed using the online STRING database 49 , using all SUMOylated proteins as input. Protein interaction enrichment was performed based on the amount of interactions in the networks, as compared to the randomly expected amount of interactions, with both variables directly derived from the STRING database output. Visualization of the interaction network was performed using Cytoscape version 3.0.2 59 .
  • SUMO target protein analysis all proteins identified in this work with at least one SUMO site were selected.
  • SUMO-2 target proteins were extracted from Becker et al. 15 , Golebiowski et al. 19 , Bruderer et al. 31 , and Schimmel et al. 48 .
  • SUMO-2 target proteins identified by site were extracted from Matic et al. 41 , Schimmel et al. 48 , and Tammsalu et al 32 . Where required, gene IDs were mapped to the corresponding Uniprot IDs. Perseus software was used to generate a complete gene list for all known human proteins, and all identified SUMO target proteins from our study as well as the above-mentioned studies were aligned based on matching Uniprot IDs.
  • PhosphoSitePlus PhosphoSitePlus®, www.phosphosite.org, 60
  • sequence windows were assigned. For each dataset, duplicate sequence windows were removed.
  • Perseus software was used to generate a matrix where all sequence windows from all PTMs were cross- referenced to each other.
  • SENPl shows a higher affinity for cleaving SUMO-1 as opposed to SUMO-2/3, but overall is less efficient than SENP2. Therefore, SENP2 was chosen as the main protease for identification of SUMO-2/3 sites.
  • ubiquitin levels were investigated, and found to be completely unaffected by the SUMO proteases (Figure 5C), and equal total protein levels were validated by Ponceau-S ( Figure 5D).
  • SNHSA sulfosuccinimidyl-acetate
  • SNHSSSB couples a biotin to the lysine, which is furthermore linked by a disulfide bridge. This then allowed for a second purification of proteins labeled by biotin, where they were previously modified by SUMO-2 (Figure 7A).
  • the efficacy of this approach was elucidated by monitoring total SUMO-2 throughout the procedure, as well as a known SUMO target protein, TRIM33.
  • the initial step included enrichment of His 10- SUMO-2, with acetylation performed on-beads.
  • TRIM33 seemed to be less efficiently purified after acetylation (Figure 7B), but total internal acetylation of the protein may have interfered with the antibody recognizing the protein.
  • SILAC SILAC
  • both “medium” and “heavy” SILAC labeling was performed, and lysates were mixed in equimolar ratio immediately after cell lysis.
  • the samples were concentrated over 100 kDa cut-off filters, specifically removing free
  • PRISM does not directly identify a site by modification on a peptide, and thus cannot benefit from the presence of reporter ions. Therefore, to further increase confidence of our dataset, overlap analysis to other SUMOylation studies was performed. PRISM-identified proteins were compared to three major studies aimed at identification of SUMOylated proteins 15 19 31 , and 58.4% were found to be previously identified by these three studies ( Figure IOC). When also including SUMOylated proteins recently identified by Schimmel et al. 48 , this overlap further increased to 72.6%. Comparatively, SUMO targets identified by Bruderer et al. and Becker et al. demonstrated significantly less overlap towards other studies. 19 SUMOylated proteins were identified by PRISM and all four aforementioned studies.
  • PRISM PRISM methodology
  • PRISM PRISM methodology
  • PRISM with high-resolution mass spectrometry, and identified nearly 400 wild-type SUMOylation sites on endogenous protein lysines, purified from HeLa cells. 37.5% adhered to the stringent [IVML]KxE consensus, and 67.9% adhered to either the KxE or [ED]xK short motifs.
  • SUMOylated proteins were found to be predominantly nuclear, and involved in chromatin remodeling, RNA splicing, transcription, and DNA repair. When compared to other SUMOylation studies, a significant overlap with PRISM- identified SUMO sites (31%) and SUMO target proteins (73%) was confirmed. We discovered one fifth of PRISM-identified SUMOylated lysines to overlap with ubiquitylation and acetylation. The observed modification of endogenous ubiquitin by wild-type SUMO-2 on lysine-63 suggests that SUMO-2 may be involved in blocking ubiquitin lysine-63 chain elongation.
  • PRISM Compared to published studies on SUMO, PRISM not only provides the ability to identify wild-type SUMOylation sites, but also identified more sites under standard growth conditions than any other study to date. Nonetheless, Tammsalu et al.
  • PRISM solves the tryptic remnant problem that has plagued the identification of endogenous SUMOylation sites. Acetylation does not suffer from this limitation, and many thousands of acetylation sites have been published following a direct purification using an anti-acetyl-lysine antibody 39 , whereas the biotin-switch method only identified 29 putative modification sites 51 . While investigation of the specific activity of the protease in question remains of interest, the fidelity of these proteases in vitro is often not directly comparable to in vivo activity of these proteases. Furthermore, PRISM is performed under fully denaturing conditions, ensuring inactivation of all endogenous proteases, and allowing complete blocking of lysines in endogenous proteins.
  • PRISM does not utilize biotinylation and subsequent purification. While this could be successful in reducing sample complexity, it does not address any potential background false positive hits resulting from incomplete acetylation of lysines. In order to address this issue, a negative dataset would have to be generated, where the protease step is skipped. Finally, leaving the lysine free after
  • deSUMOylation allows for identification of two reporter peptides, due to trypsin being able to cleave the peptide, with both reporter peptides being shorter and thus easier to resolve. This is especially pivotal in the lysine-blocking context, already resulting in peptides that are on average twice as long as from a non-blocked tryptic digest.
  • PRISM allows for identification of SUMOylated peptides that are lysine-rich up to the point where they would normally be unidentifiable due to being too short.
  • PRISM can be utilized in a wider context to chart more wild-type SUMOylation sites in endogenous proteins, by investigation of different cell lines and in response to varying stimuli.
  • the methodology is generic and is therefore widely applicable to study lysine post-translational modifications.
  • PRISM can be used to characterize wild-type SUMO sites in highly complex in vitro and in vivo samples.
  • SUM02 is essential while SUM03 is dispensable for mouse embryonic development. EMBO Rep. 2014.
  • RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat. Cell Biol. 2008, 10 (5), 538-546.
  • Dominguez-Vidana R.; Liang, A. C; Solimini, N. L.; Bernardi, R. J.; Yu, B.; Hsu, T.; Golding, I.; Luo, J.; Osborne, C. K.; Creighton, C. J.; Hilsenbeck, S. G.; Schiff, R.; Shaw, C. A.; Elledge, S. J.; Westbrook, T. F. A SUMOylation-dependent

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Abstract

La présente invention concerne des procédés pour détecter des lysines ayant subi une modification post-traductionnelle dans un polypeptide. Un type spécifique de modification post-traductionnelle des lysines peut être mis en correspondance au niveau du protéome. En outre, l'invention concerne un procédé permettant de détecter si une protéine contient une modification post-traductionnelle protéique.
PCT/NL2014/050581 2013-08-27 2014-08-27 Procédés pour détecter des lysines ayant subi une modification post-traductionnelle dans un polypeptide WO2015030585A2 (fr)

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US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
CN109557193A (zh) * 2018-07-09 2019-04-02 中国检验检疫科学研究院 一种芝麻主要过敏原的质谱定性检测方法
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