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Oded Kleifeld

Alain Doucet

Jayachandran N. Kizhakkedathu

Christopher M. Overall

Figure 1

System-wide proteomic identification of protease cleavage products by terminal amine isotopic labeling of substrates

The sequence and nature of all the protein amino-termini \(N-termini) within the proteome \(the N-terminome) provides valuable functional annotation, since translation start sites, N-terminal isoforms, modifications and truncations determine the cellular localization, activity and fate of most proteins1. As ==~== 85% of eukaryotic proteins have an acetylated N-terminus2 and all proteins undergo proteolysis3, these are not only two of the most ubiquitous, but also two of the most important post-translational modifications4,5. The protein amino-terminus is susceptible to amino-terminal peptidase processing, modification of the alpha-amino group, and side-chain specific changes that can target a protein for ubiquitination and degradation or protect it from rapid turnover and so determines its half-life1. In addition to constitutive proteolysis, regulated processing of protein amino termini can irreversibly change the protein activity or properties6-8 but the extant to which proteolysis sculpts the proteome is unknown4. Hence, it is important to determine the cleavage site within each protease substrate, since the biological activity of the cleavage products is commonly determined by the precise fragmentation pattern.


 


With 569 members, proteases are the second largest enzyme class in man9 and are 5-10% of drug targets10. Crucial to linking a specific protease with a defined biological pathway and for drug development is determining the substrate repertoire, or substrate degradome11, of a protease since this can generate hypotheses on its role and provide biomarkers of drug efficacy. Yet, for around half of the proteases in man no substrates are known and for the other half, the substrate degradome is incompletely annotated3,11.Thus specific degradomics techniques are needed to rapidly identify and quantify the N-terminome in order to reveal the extent of proteolysis in a system, the functional state of key molecules, and to identify new substrates.





Positional proteomics approaches that isolate only the N-terminal peptides of proteins, the N-terminome12-16, have been proposed for sample simplification before mass spectrometric \(MS) analysis and for proteome annotation, but coverage is often limited12-15,17,18 and aside from combined fractional diagonal chromatography \(COFRADIC) most of these approaches were not reported for global protease cleavage site analysis. The main obstacles towards this latter goal are the identification the neo-N-termini of the substrates generated by specific proteolysis and to distinguish these not only from the natural N-termini, but also from N-termini generated by background proteolysis of the proteins in a sample and by trypsin digestion \(internal tryptic peptides) in proteomic workflows3,19. Solving these problems requires innovative strategies to circumnavigate the very similar chemical properties of the primary amines of the lysine side chains and N-termini. Recent reports of different approaches to tackle this difficult task include lysine-specific blocking of intact proteins to expose only amino-termini for biotinylation followed by affinity capture20, specific labeling followed by _in silico_ selection of protease generated neo-N-terminal peptides21, specific enzyme-mediated biotinylation of unblocked alpha-amine groups with enrichment for the biotinylated N-terminal peptides after one reaction16. Although these approaches represent a welcome step forward for studying protease-generated neo-N-terminal peptides, they are still limited in different aspects such as quantification20, coverage21 or are potentially biased16 and more importantly incapable of analysing naturally blocked N-terminal peptides. To date, COFRADIC is the only N-terminomics approach that provides, broad coverage and isotopic quantification and can be applied to study protease substrate degradomes as well as to completely annotate the N-terminome12,22-25. However COFRADIC is an expensive and time-consuming procedure involving multiple and complicated enzymatic and chemical steps, multiple HPLC fractionations and up to 150 MS/MS analyses per experiment25.





To overcome these problems we developed a new positional proteomics approach: Terminal Amine Isotopic Labeling of Substrates \(TAILS)26. TAILS is a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative analysis of the N-terminome and proteolysis on a proteome-wide scale in one MS/MS analysis. By a three-day procedure with flexible labeling options, TAILS removes internal tryptic and C-terminal peptides to enrich for all forms of N-terminal peptides by negative selection. To overcome nonspecific peptide binding and the low capacity of derivatized chromatographic beads that in some techniques necessitates large sample amounts for analysis, we developed a novel class of dendritic polyglycerol aldehyde polymers optimized for efficient, high capacity tryptic peptide binding with virtually no non-specific interactions. Rather than deliberately excluding acetylated proteins13,16,20,21, TAILS provides wide coverage of all forms of naturally blocked N-terminal peptides and allows for their quantification through isotopic labeling of lysine side-chains. In addition to annotating the proteome we utilze these peptides to form a statistical classifier for the TAILS experiments to determine statistically valid isotope ratio cutoffs.

**Cell lines**


Cell lines from an organism with a fully sequenced genome.





**Reagents**


These should be of the purest available:


10% sodium dodecyl sulfate polyacrylamie gel electrophoresis \(SDS-PAGE) gel plus loading and running buffers and silver stain solutions.


Acetone - \(Sigma-Aldrich).


Acetonitrile - \(Sigma-Aldrich).


Dithiothreitol \(DTT) - \(Sigma-Aldrich).


Ethylenediaminetetraacetic acid \(EDTA) - \(Sigma-Aldrich).


12CH2-formaldehyde \(CH2O) - 12.3 M for light labeling \(37% \(vol/vol), Sigma-Aldrich, cat. no. "252549":http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&amp;N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&amp;N4=252549%7CSIAL&amp;N25=0&amp;QS=ON&amp;F=SPEC).


13C2H2-formaldehyde \(13CD2O) - 6.6 M for heavy labeling \(20% in D2O, 99% 13C, 98% D, Cambridge Isotopes, cat. no. "CDLM-4599-1":http://www.isotope.com/cil/products/displayproduct.cfm?prod_id=4208) formaldehyde vapors are toxic - prepare solutions in a fume hood.


Formic acid \(Sigma Aldrich).


Guanidine hydrochloride \(GuHCl) - \(Sigma-Aldrich).


4-\(2-hydroxyethyl)piperazine-1-ethanesulfonic acid \(HEPES) \(Sigma-Aldrich), 1 M pH 7.0.


High molecular weight-aldehyde derivatized \(HPG-ALD) polymer at around 35 mg/ml.


_Note_: "HPG-ALD":http://www.flintbox.ca/technology.asp?page=3081 polymers for proteomics are available through Flintbox, The Global Intellectual Exchange and Innovation Network - "www.flintbox.ca":www.flintbox.ca , Flintbox Innovation Network Inc., 21 Water Street, 5th Floor, Vancouver BC, Canada, V6B 1A1.


Iodoacetamide \(IAA) - \(Sigma-Aldrich), 0.5 M stock in water.


_Note_: Iodoacetamide stock solution should be freshly prepared and kept at 4 °C.


Methanol \(Sigma-Aldrich).


Sodium chloride \(NaCl) - \(Sigma-Aldrich).


Sodium cyanoborohydride \(NaBH3CN) 1 M \(ALD coupling solution, Sterogene, cat. no. "9704-01":http://www.sterogene.com/product_detail.php?ID=9704-01).


_Note_: ALD solution should be kept at 4 °C and not stored past the expiry date to ensure good labeling efficiency.


Phenylmethylsulphonyl fluoride \(Sigma-Aldrich), 100 mM stock \(100x).


_Caution_: PMSF is carcinogenic. The stock solution in ethanol or acetonitrile should be freshly prepared.


Phosphate buffered saline \(PBS), sterile \(138 mM NaCl, 2.7 mM KCl, 20 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4).


Protein concentration assay.


Test protease for TAILS assay.


Trifluoroacetic acid \(TFA) \(Sigma-Aldrich).


Trypsin, mass spectrometry grade \(Promega).


Microcapillary tubes \(Hamilton).

1.5 mL microfuge tubes  \(e.g. Eppendorf or equivalent).


_Note_: Polymers released from tubes and surfaces upon exposure to chemicals and solvents contaminate and interfere with mass spectrometric analysis. We have found that microtubes from Eppendorf show good chemical resistance and are suitable for the procedures described in this protocol.


15 mL polyethylene sterile tubes \(e.g. "Nunc":http://www.nuncbrand.com/us/page.aspx?ID=10558, "Corning":http://catalog2.corning.com/Lifesciences/en-US/Shopping/ProductDetails.aspx?productid=430052%28Lifesciences%29&amp;categoryname=General+Labware+and+Equipment%28Lifesciences%29%7CCentrifuge+Tubes+%28Lifesciences%29%7CCentrifuge+Tubes%2c+Disposable+Plastic+%28Lifesciences%29%7C15mL+Screw+Cap+Conical+Bottom+Centrifuge+Tubes%28Lifesciences%29, or equivalent).


_Note_: These tubes are going to be used for acetone precipitation, thus they require chemical resistance to acetone and methanol, and centrifugation at 15,000g.


50 mL tubes for collection of cell-conditioned medium.


50 mL 0.22 &#x3BC;M filtering devices such as Millipore "Steriflip":http://www.millipore.com/catalogue/module/c3238 or equivalent.


Argon gas.


Centrifugal protein concentrator with 5-kDa molecular weight cut-off such as Millipore "Amicon-Ultra 15":http://www.millipore.com/catalogue/module/c7715 concentrator.


Centrifuge for volumes 50 ml tubes at up to 20,000g.


Computer equipped with proteomic analysis software.


Dialysis equipment with a 10-kDa molecular weight cut-off \(the Pierce "Slide-A-Lyzer":http://www.piercenet.com/Products/Browse.cfm?fldID=293902DB-5056-8A76-4E73-286D1FC1B2A5#minidialysisunits works well).


Standard laboratory equipment for cell culture, molecular biology, and protein chemistry.


Reversed-phase solid phase extraction cartridges \(e.g. Waters "Sep-Pak C18":http://www.waters.com/waters/partDetail.htm?locale=en_US&amp;partNumber=WAT023501 light or equivalent).


Millipore "Microcon":http://www.millipore.com/catalogue/item/679 spin-filter device with a 30-kDa molecular weight cut-off.


LC-MS/MS instrument.


Liquid nitrogen.


pH test strips range 6-9 \(e.g. from "Merck":http://www.emd-chemicals.ca/ph-indicator-strips-non-bleeding/c_ViKb.s1L.CAAAAEWmOIfVhTl).


Protein concentration determination assay.


SDS-PAGE apparatus and power supply.


Table-top centrifuge accommodating 2 mL reaction tubes.


Vacuum evaporation system \(commonly referred to as a "speed-vac").

Highly variable depending on cell lines and data analysis time. About 5 days including a 24 h cell culture time in serum-free media.

Do not use buffers or reagents with primary amines prior to completion of labeling and the last step of polymer negative selection.


Monitor and adjust pH when indicated.


Cool samples to room temperature before alkylating cysteine with IAA.


Do not use urea for sample resolubilization.


Quench completely excess IAA with DTT.


Quench completely excess labeling reagent with ammonium bicarbonate prior to mixing heavy and light labeled samples and tryptic digest.


Block polymer functional groups with ammonium bicarbonate after completion of binding of the internal tryptic and C-terminal peptides to the polymer in the negative selection step of blocked peptides.


Perform all labeling steps in fume hood.

TAILS is a highly optimized procedure. Many of the steps are performed to be mass spectrometer compatible at later stages. Do not vary the parameters and conditions until satisfactory results are obtained. 





If very few peptides are identified by the database search:


Verify the quality of the MS data \(mzXML) using "Pep3D":http://tools.proteomecenter.org/wiki/index.php?title=Software:Pep3D.


Change database search parameters to include potential modifications.


Validate the tested protease activity \(as suggested in "Test protease cleavage of collected proteome").


Increase the protease:proteome ratio or incubation time.


Verify labeling by analyzing initial labeled samples \(subsection 19 in "isotopic labeling").


Use protease of known specificity such as GluC or caspase to set the analysis conditions before using the test protease.

Anywhere from 100 to 1000s of cleavage sites for the tested protease and ==~== 100-1000 original free and blocked N-terminal will be identified. This depends on the size and complexity of the tested proteome and activity and presence of substrates of the test protease.

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O.K. was supported by the Centre for Blood Research \(CBR) \(University of British Columbia), Canadian Institute for Health Research/Heart and Stroke Foundation of Canada \(CIHR/HSFC) Strategic Training Program in Transfusion Science research fellowship. A.D. acknowledges the Fonds Quebecois de la Recherche sur la Nature et les Technologies and the Michael Smith Foundation for Health Research \(MSFHR) for research fellowships. J.N.K. is the recipient of a Canadian Blood Services \(CBS)/CIHR new investigator award in transfusion science. C.M.O is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. This work was supported by grants from the CIHR and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance with funds from the Canadian Breast Cancer Foundation and The Cancer Research Society as well as with an Infrastructure Grant from the Canada Foundation for Innovation \(CFI) and MSHFR. We thank Dr Wei Chen and the CBR mass spectrometry core facility for proteomics analysis and Dr Georgina Butler for critical reading of the manuscript.

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Method Article

Oded Kleifeld, Alain Doucet, Jayachandran N. Kizhakkedathu, Christopher M. Overall

Oded Kleifeld

Israel Institute of Technology, Faculty of Biology, Haifa 32000, Israel.

Alain Doucet

Department of Biochemistry and Molecular Biology, University of British Columbia.

Jayachandran N. Kizhakkedathu

Department of Pathology and Laboratory Medicine, University of British Columbia.

Christopher M. Overall

Department of Oral Biological and Medical Sciences, University of British Columbia

DOI:

10.1038/nprot.2010.30

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Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products

by Oded Kleifeld, Alain Doucet, Ulrich auf dem Keller, +6

Nature Biotechnology (11 February, 2010)

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Method Article

Oded Kleifeld, Alain Doucet, Jayachandran N. Kizhakkedathu, Christopher M. Overall

Oded Kleifeld

Israel Institute of Technology, Faculty of Biology, Haifa 32000, Israel.

Alain Doucet

Department of Biochemistry and Molecular Biology, University of British Columbia.

Jayachandran N. Kizhakkedathu

Department of Pathology and Laboratory Medicine, University of British Columbia.

Christopher M. Overall

Department of Oral Biological and Medical Sciences, University of British Columbia

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DOI:

10.1038/nprot.2010.30

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