Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to

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Immuno-affinity Capture followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins Majlinda Kullolli, Dan A Rock, and Ji Ma J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00863 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 20, 2016

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Journal of Proteome Research

Immuno-affinity Capture followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins

Majlinda Kullolli *1 , Dan A. Rock 1 , and Ji Ma 1 1

Department of Pharmacokinetics and Drug Metabolism, Amgen Inc.,

South San Francisco, CA 94080, USA Corresponding authors email: [email protected]

Corresponding author: Dr. Majlinda Kullolli 1120 Veterans Blvd Amgen Inc. South San Francisco CA 94080, USA

KEYWORDS N-succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl) phosphonium, proteolytic degradation, on-bead tagging, immunoprecipitation

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ABSTRACT Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization mass spectrometry (ESI-MS) analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the Nterminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent LC-MS/MS. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTshuFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.


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INTRODUCTION Since the development of recombinant DNA methodology and genetic engineering from the late 1970s, therapeutic proteins have increasingly become a critical tool for treating diseases.1 While monoclonal antibodies account for majority of currently approved protein drugs, recent advancement of protein engineering technologies have provided many novel therapeutic protein platforms, including fusion proteins, PEGylated proteins, various antibody fragment formats and antibody-drug conjugates (ADC). These genetically and/or chemically engineered molecules afforded exciting options for development of therapies with optimized pharmacological, pharmacokinetic and toxicological profiles.2, 3 In contrast to small molecule drug discovery process, understanding of absorption, disposition, metabolism, and excretion (ADME) profiles of therapeutic proteins, primarily monoclonal antibodies, have been limited due to their often exquisite properties; e.g. high bioavailability and low clearance.4, 5 However, many novel therapeutic protein modalities possess unique structural and conformational diversities relative to antibodies that result in an increase in ADME liabilities including fast proteolytic degradation that could lead to impaired pharmacokinetic, pharmacological and safety profiles.4 Thus, early detection of specific sites of proteolytic cleavage has become critical to engineering stable therapeutic candidates.4-8 In addition, understanding the metabolic liabilities of therapeutic candidate will ensure proper selection of reagents for ligand binding assay (LBA); further will ensure appropriate assessment of pharmacological and toxicological effects.8, 9 Liquid chromatography – mass spectrometery (LC-MS) based techniques are primarily used to characterize therapeutic protein catabolism. A typical experiment consists of immunoaffinity



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capture (IC) of the desired protein, followed by top-down analysis of the protein using either MALDI-MS or ESI-MS.8, 10, 11 Even with these efforts immunoaffinity capture of the desired protein can result in complex sample mixtures. The multiple stages of sample treatment that include enrichment, washing, concentration, and proteolysis can introduce significant sample losses. Therefore, top-down MS analysis of such a sample mixture using either MALDI or ESI MS can be challenging. Alternatively, therapeutic protein catabolism can be studied through targeted LC-MS/MS where the therapeutic protein is subjected to a protease and a specific proteolytic peptide is monitored using multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer (QQQ).12 This methodology is sensitive and specific, but prior knowledge of the proteolytic cleavage site through surrogate experiments is a requirement. N-terminus tagging followed by enzyme digestion and LC-MS/MS analysis has been one of the common bottom-up protein mass spectrometry approaches in many applications, e.g. protein characterization, global proteomics analysis. Among many N-terminus tags that have been developed, succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl) phosphonium (TMPPAc-OSu) has emerged as a promising new reagent. TMPP-Ac-OSu has been applied successfully to many different applications such as de novo N-terminal peptide sequencing, oriented proteogenomics, and as well as to accurate quantitation of the protein by using with light/heavy TMPP-labeling of the peptide.3, 12-16 The intrinsic hydrophobicity of the tag allows a later elution of the N-terminus peptides in a reverse phase LC run. This not only facilitates better separation and subsequent easier identification of the N-terminus peptide but also improves the ionization efficiency in the presence of higher percentage of organic solvent in the eluent. The extra positive charge that the TMPP tag carries further enhances the ionization efficiency, promotes 4 ACS Paragon Plus Environment

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formation of multi charge species, and enhances collision induced dissociation efficiency of the peptide species. Experimentally, TMPP tag is fully compatible with most common reagents used in bottom-up proteomic workflows, resulting in less sample manipulation prior to LC-MS analysis. Herein a TMPP N-terminus tagging (TMPP-NTT) based bottom up proteomics workflow was designed and evaluated to study therapeutic protein catabolism. This workflow consists of enrichment of therapeutic protein and its catabolite by immunoaffinity capture using streptavidin magnetic beads coated with biotinylated anti human Fc antibody, followed by on-bead Nterminus tagging. The eluted tagged proteins were subsequently digested and analyzed by nanoflow LC-High Resolution mass spectrometery (HRMS). Analysis of the MS/MS data allowed definitive sequence identification of all N-terminus peptides related to therapeutic protein and its catabolites; and such information can lead to unambiguous identification of proteolytic degradation sites in the therapeutic protein. As a proof of concept, we applied this workflow to study in vitro catabolism of neurotensin-huFC (NTs-huFc) fusion protein in mouse serum.



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EXPERIMENTAL SECTION Reagents: Succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl) phosphonium (TMPPAc-OSu) was purchased from Sigma Aldrich (St. Louis, MO). The following items were purchased from ThermoFisher Scientific (Carlsbad, CA): NuPAGE® MES SDS running buffer, 4-12% Bis-Tris Gel, NuPAGE LDS 4x sample buffer, and Dynabeads Streptavidin M280. HPLC grade acetonitrile, HPLC grade water, urea, thiourea, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS)-polyacrylamide, ammonium biocarbonate (NH4HCO3 ) , iodoacetamide (IAA), dithiothreitol (DTT), phosphate-buffered saline (PBS), sodium chloride (NaCl), guanidine chloride (GuCl) and sodium acetate were purchased from ThermoFisher Scientific (Waltham, MA ). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). Tributylphosphine was purchased from BioRad Laboratories (Hercules, CA). Mouse serum was purchased from Bioreclamation Inc (Long Island, NY). Biotinylated antihuman Fc antibody (biotinylated Ab35) was prepared by Amgen Inc (Thousand Oaks, CA). In Solution TMPP-NTT of NTs-huFc Fusion Protein: A previously published in-gel digestion protocol was followed,15 the neurotensin huFc fusion protein (1 µg) was denatured with 50 mM Tris, 8 M urea, 2 M thiourea, 1 mM EDTA, 5 mM , and 5 mM tributylphosphine. A solution of 0.1 M TMPP-Ac-OSu in CH3CN:water (2:8, v/v) was prepared and added to the denatured protein at a molar ratio of 200:1. The mixture was incubated at room temperature for 60 min under gentle agitation. The excessive TMPP reagent was quenched by adding hydroxylamine to a final concentration of 0.1 M, followed by incubation at room temperature for 60 min under gentle agitation. To remove the quenched TMPP reagent the samples were run on 4-12% SDS PAGE gel for 45 min at 200 V. The gel was stained using Coomassie blue staining. The gel was destained thoroughly (overnight), to a clear background and nicely visualized bands. After 6 ACS Paragon Plus Environment

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protein separation based on size the bands of interests were excised from the gel. Previously published in-gel digestion protocol was followed.17 Briefly, the gel slices containing the bands of interest were dehydrated with CH3CN followed by hydration with 25 mM NH4HCO3 (this step was repeated three times). The disulfide bonds of the proteins were subsequently reduced through incubation with 10 mM of DTT at 56 °C for 30 min. After cooled down to room temperature, the reduced protein samples were alkylated with 55 mM IAA followed by incubation for 20 min at room temperature in dark. After dehydration with acetonitrile, the liquid was removed and the gel pieces were submerged in trypsin solution (13 ng/µL), and incubated for 2 hr in 4 °C. To ensure that the gel pieces were saturated with trypsin solution, a second aliquot of trypsin solution was added to the samples. Following the addition of the second aliquot of trypsin solution, the samples were then incubated overnight at 37 °C in a thermomixer shaking at 800 rpm. The peptides were then extracted using 60% acetonitrile aqueous solution containing 5% formic acid. The volumes of extracted peptides samples were concentrated down to 10 µL each using SpeedVac concentrator, prior to LC-MS/MS analysis. TMPP N-terminus Tagging Selectivity of NTs-huFc Fusion Protein. To evaluate the impact of pH condition in the reaction buffer to the N-terminus tagging selectivity, following previously described method, the TMPP tagging reaction of NTs-huFC fusion protein (150 ng per sample) was carried out at the pH conditions of 6.4, 7.4, and 8.2. The resulted samples were analyzed by the same LC-MS/MS methods. Immonoaffinity Capture of NTs-huFC Fusion Protein from Mouse Serum: The immunoaffinity capture process was carried out using 300 µL of Dynabeads streptavidin magnetic beads M280 (10 mg/mL) crosslinked with biotinylated anti-human Fc monoclonal antibody (b-Ab35, Amgen). In a typical experiment, the b-Ab35 (4.1 mg/mL) was added to 7 ACS Paragon Plus Environment


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streptavidin beads at ratio of 1:5 and incubated overnight at 4 °C under end-to-end rotation. The biotinylated-b-Ab35-crosslinked streptavidin magnetic beads were then washed three times with 1 mL each of 10 mM HEPES containing 300 mM NaCl at pH 7.2. For the immunoaffinity capture of NTs-huFc fusion protein, 50 µL of b-Ab35-crosslinked beads was added to 200 µL of mouse serum spiked with 200 ng of the protein (the mouse serum was diluted with 600 µL of 10 mM Hepes, 300 mM NaCl at pH 7.2), and subsequently incubated for 4 hr at 4 °C under end-toend rotation. After incubation, streptavidin magnetic beads washes and elution steps were conducted using the KingFisher magnetic particle processor (Thermo Fisher Scientific, San Jose, CA). Following transfer of the magnetic beads-serum mixture to a 96 well plate, the magnetic beads were pulled from the serum solution, and subsequently washed with 1 mL of 10 mM Hepes containing 300 mM NaCl at pH 7.2 for 5 cycles at 90 seconds each. The beads were then washed two more times with 1 mL of PBS using the same cycle time settings. Based on different workflows, the cleaned beads were released to next step, including elution of the protein samples, or direct onbead digestion. Workflow 1: In-solution Tagging Followed by In-gel Digestion: Using the KingFisher device, the immunoaffinity captured protein samples were eluted from magnetic beads using 100 µL of 100 mM sodium acetate at pH 3.6. The eluent was concentrated down to 25 µL using a SpeedVac concentrator. TMPP tagging of the protein samples was carried out using the insolution tagging protocol described previously, followed by in-gel sample clean up and digestion prior to LC-MS/MS analysis.


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Workflow 2: On-bead Tagging Followed by On-Bead Digestion: Following the enrichment of the NTs-huFC protein by the streptavidin magnetic beads crosslinked with b-Ab35, the TMPP tagging reaction was performed without protein elution from the beads. Continuing from the previous described KingFisher based immunoaffinity capture process, the beads were released into a 50 mM Tris-HCl buffer at pH 8.2 containing TMPP-Ac-OSu at an estimated ratio of 200 to 1. The mixture was incubated at room temperature for 60 min under gentle agitation. The excessive TMPP reagent was then quenched by adding hydroxylamine to a final concentration of 0.1 M, followed by incubation at room temperature for 60 min under gentle agitation. Removal of the quenched excessive TMPP was performed using the KingFisher device. Briefly, the beads mixture was washed with three times using 1 mL each of PBS buffer for 5 cycles at 90 seconds each. The washed beads were then released into 80 µL of reduction buffer containing 30 µL PBS, 45 µL of 8 M urea, and 10 mM DTT, followed by incubation at 56 °C for 30 min in a thermomixer shaking at 800 rpm. The mixture was then let to cool down prior to the 45 min alkylation reaction using 25 mM IAA. Dilution of the samples with 100 mM ammonium bicarbonate at pH 8.0 allowed reduction of for the urea concentration to be less than 1 M. Typically digestion of the sample was initiated by addition of 10 ng of trypsin, followed by overnight at 37 °C. The reaction was then quenched by adding 4 µL of 10 % formic acid aqueous solution. After removal of the magnetic beads, the supernatant was further cleaned up by SPE using Waters HLB µelution plates. The SPE eluent were then concentrated down and subjected to LC-MS/MS analysis. Workflow 3: On-bead Tagging Followed by In-Solution Digestion As a modification from the workflow 2, after the on-bead TMPP tagging reaction and the follow up clean up steps, the tagged protein samples was eluted from the beads using 100 µL of 100 mM sodium acetate at pH 9 ACS Paragon Plus Environment


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3.6. The sample was immediately neutralized with 10% Tris-HCl buffer at pH 8.0, and subsequently concentrated down to 10 µL. In-solution trypsin digestion of the TMPP tagged proteins was performed using previously described method.18 Briefly, samples were denatured with addition of 5 volumes of 6.0 M GuCl in 0.1 M ammonium bicarbonate solution at pH 8.0. The disulfide bond reduction was achieved by addition of 10 mM DTT and incubation at 56 °C for 30 min. The reduced proteins were let to cool down, and alkylated with 25 mM IAA for 30 min at room temperature in the dark. Following 5 mM DDT reaction quench, the samples were diluted with 100 mM ammonium bicarbonate buffer at pH 8.0 to reduce the guanidine-HCl concentration down to 0.5 M. Proteins were then digested with trypsin at an enzyme to protein ratio of 1:40 (w/w). The digestion reaction was terminated by the addition of formic acid to a final concentration of 1%. The tryptic digestion samples were then subjected to LC-MS/MS analysis. To assess the tagging consistency of the workflow, three independent experiments were performed using workflow 3. In Vitro Catabolic Stability Study of NTs-huFC Fusion Protein in Mouse Serum: The protein was incubated at 37 °C in mouse serum at a concentration of 1 µg/mL. A total of 4 time points were collected over a period of 120 min incubation. The incubation samples were processed accordingly using the workflow 3 prior to LC-MS/MS analysis. LC-HRMS Method: LC-MS/MS analyses of TMPP tagged protein digestion samples were performed on a nanoACQUITY M-Class UPLCsystem (Wates, Manchester, UK) coupled with high resolution Fusion Tribrid orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). In a typical experiment, following injection of 10 µL of protein digestion sample, the tryptic peptides were trapped with a Symmetry C18 trap column (5µm x 200 mm x 0.18 mm) (Waters, Milford, MA), and subsequent separation was achieved using a 1.7 µm particle size 10 ACS Paragon Plus Environment

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ACQUITY UHPLC BEH column (200 mm x 75 µm) (Waters, Milford, MA). The mobile phase system consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). Post sample injection, peptide trapping was performed for 10 min at a flow rate of 5 µL/min with 5% mobile phase B. Separation of these tryptic peptides was performed at a flow rate of 0.3 µL/min, using a linear gradient 5-60% mobile phase B over 40 min; after which 90% mobile phase B was maintained for 5 min before the column was reconditioned with 5% mobile phase B for 5 min. The orbitrap mass spectrometer was operated in positive electrospray ionization mode using the following conditions: capillary voltage at 2.3 kV, ion transfer tube temperature at 275 ͦ C. The normalized collision energy was at 35% for all data dependent MS/MS scans. A top 7 data dependent MS/MS method was used for all the sample analysis. Following a full MS scan over the mass range of m/z 400 to 2000 using the resolution of 120,000 at m/z 200, data dependent CID MS/MS linear ion trap scans were performed on the top 7 precursor ions based on ion intensity. Dynamic exclusion was continued for duration of 30 seconds. MS Data Analysis and Identification of Proteolytic Degradation Site: Based on full MS scan and MS/MS data, peptide identification and subsequent peptide mapping analysis was performed through a customized database search against the NTs-huFC fusion protein using Proteome Discoverer 1.4 (version 1.4.0.288, Thermo Fisher Scientific, San Jose, CA). In a typical search, the mass tolerance for precursor ion was set at 10 ppm, and a 0.06 Da tolerance was set for the product ions. Full trypsin enzyme specificity was fixed with maximum two missed cleavages allowed. Carbamidomethylation of Cysteine (+57) residues was defined as fixed modification while Methionine oxidation (+16), N-terminal cyclization of Glutamine to pyro Glutamine (-17), and N-terminus TMPP (+572.18 Da) derivatization were searched as variable modifications. 11 ACS Paragon Plus Environment


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MS/MS spectra yielding a positive identification of TMPP tagged peptide were inspected manually. In order to minimize the level of false positive identifications, criteria that would yield an overall confidence of over 95% for peptide identification were established for filtering raw peptide identifications. This was achieved by filtering the Proteome Discoverer results using the following criteria, Xcorr 1.6, 1.9, and 2.3 for singly, doubly, and triply charged ions, respectively. False positive rate was set at 1% at both peptide and protein level. In addition to confirmation of N-terminus peptide of the parent NTs-huFc fusion protein, additional N-terminus TMPP tagged peptides identified in the MS data analysis led to identification of proteolytic degradation sites.


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RESULTS AND DISCUSSION Identification of TMPP Tagged Peptides in the Stock Solution. A previously published TMPP N-terminus tagging protocol had been successfully applied to the complete proteome study of Mycobacterium genus.19 To evaluate the ability to extend this protocol to characterize N-terminus derived proteolytic clips, neurotensin human Fc fusion protein (NTs-huFc) was used as a test molecule. NTs-huFc fusion protein was generated by fusing a biologically active neurotensin peptide to the N-terminus of Fc domain of human immunoglobulin G1 via G4S linker (Figure 1). Purified NTs-huFc protein was labeled using the method described above, which utilized in-solution TMPP tagging followed by removal of excessive TMPP reagent with one dimensional SDS-PAGE, and in-gel digestion of the bands of interest. The extracted tryptic peptides were then analyzed by LC-MS/MS. Following this protocol, greater than 85% sequence coverage was achieved. Identification of N-terminus peptides was based on the characteristic 572.18 Da mass tag from the TMPP group. As shown in Table 1, a total of three TMPP Nterminus tagged peptides were identified in the sample. In addition to the N-terminus peptide QLYENKPR, two other tagged peptides identified are CLP1 (AKGQPR) and CLP2 (CKVSNK). These two semi-tryptic peptides led to discovery of pre-existing clipping products in the stock solution of the NTs-huFc fusion protein. However, low ion intensity and limited MS/MS spectral fragments suggested low abundance of these degradants in the sample. As shown in Figure 2a, addition of the hydrophobic TMPP tag led to approximately 20 min longer retention time relative to corresponding untagged peptides. The untagged N-terminus peptide is identified at retention time of 20.82 min (peak 1, doubly charged, m/z 515.7697), whereas the tagged N-terminus peptide is identified at retention time of 46.37 min (peak 4, triple charged, m/z 534.9095). Due to the hydrophobic character of the TMPP, CLP1 and CLP2, are 13 ACS Paragon Plus Environment


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short peptides which makes them difficult to be retained in a HPLC column, thus these two peptides were identified only with the TMPP tag Figure 2a (peak 2 and 3), the MS/MS fragmentations of these peptides are presented in Figure 2e and 2d, respectively. The addition of the positive charge from the TMPP tag generated triply charged ions as primary charge state for these peptides. The added charge states improved CID MS/MS ionization efficiency and also led to the formation of more y- and b- ions that were critical to definitive peptide identification (Figure 2b and 2c). For NTs-huFc fusion protein, the untagged N-terminus peptide (QLYENKPR) formed predominantly doubly charged ions with m/z of 515.7698; whereas the corresponding TMPP tagged N-terminus peptide generated primarily triply charged species with m/z of 534.9095 (Figure 2a). As shown in Figure 2a, detection of untagged N-terminus peptide suggests incomplete tagging using the in-gel digestion method. Even though the TMPP is a relatively small molecule, fully unfolded protein is critical to ensure ready access to the Nterminus of the structure. Thus, one can hypothesize that incomplete protein denaturation could led to incomplete labeling of the N-terminus peptide with TMPP using this method. TMPP N-terminus Tagging Selectivity. For TMPP tagging to be utilized effectively for identification of proteolytic instability the tagging process must be selective for the N-terminal residue. To investigate the specificity of TMPP tagging towards N-terminus primary amine of the NTs-huFc fusion protein, the same in-solution tagging and in-gel TMPP removal and digestion method was used with the exception of pH condition of the TMPP reaction buffer (Table 2). While at the regular pH of 8.2, three tagged peptides were readily detected, no TMPP was tagged to the target proteins when the pH was the reaction buffer was lowered to 6.4 and 7.4. In addition, at pH 8.2, a non-selective TMPP tagged peptide (RPYILGGGGSDK) was observed. The untagged peptide triple charged at m/z of 407.2204, whereas the corresponding 14 ACS Paragon Plus Environment

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tagged peptide +4 m/z of 448.7123 were both identified, however the tagged peptide had significantly lower abundance than the tagged peptide as shown in Figure 3a and its corresponding MS/MS fragmentation shown in Figure 3b and 3c. While this was an isolated incident in all experiments carried out in the study, the identification of the non-selective TMPP tagged peptide suggests careful control of reaction condition is crucial to maintain N-terminal tagging selectivity. Development of Immunoaffinity Capture Followed by On-Bead TMPP-NTT Workflow. A TMPP N-terminus tagged peptide based workflow has been designed and optimized to meet the sensitivity, robustness and throughput needs for in vitro and in vivo catabolism studies of therapeutic proteins. In the TMPP tagging process, total removal of excess quenched TMMP prior to LC-MS analysis is required to allow generation of quality and reproducible data as co-elution of free TMPP reagent with tagged peptides significantly reduced ionization and CID fragmentation efficiency. Membrane filtration devices were previously used to remove the excess TMPP with limited success; significant sample losses were also observed compared to 1D SDS-PAGE separation method.19 The 1D SDS-PAGE has been the primary method of choice for removal of excess TMPP reagent; however it is labor intensive and requires excessive many sample manipulation steps that can easily lead to large intra- and inter-assay variability. Another point of consideration in the design of the workflow was efficiency and reproducibility of tryptic digestion process that has significant impact on applicability of the approach to the intended purpose. In this study, three different methods were evaluated for incorporation of the TMPP tagging methodology to the workflow (Figure 4).



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Workflow 1: In-solution Tagging Followed by In-gel Digestion: As shown in Figure 4, after immunoaffinity capture of NTs-huFc fusion protein from mouse serum sample, the common insolution tagging followed by in-gel digestion method was used to generate the TMPP tagged peptides prior to LC-MS/MS analysis. Based upon this methodology good sequence coverage of the target protein was achieved as shown in Table 3. However, the tagged TMPP N-terminus peptide was not identified using this workflow. Instead, only the untagged N-terminus peptide was identified with low abundance. This observation could suggest stronger retention of the TMPP tagged peptide in the gel. While this could be possible previous efforts with in-gel isolation of TMPP tagged peptides are inconsistent with this hypothesis. Alternatively, it could be that incomplete tagging efficiency occurred using this workflow. While this common in-gel method allowed complete removal of excess quenched TMPP reagent and ready-to-inject samples without additional steps, multiple sample manipulation steps over the course of several days not only increase the possibility for sample losses, but also limits its application for routine rapid studies. Furthermore, incomplete digestion could be due to poor access of trypsin to the target protein in the gel matrix followed by inefficient extraction of the digested peptides from the gel. Workflow 2: On-bead Tagging Followed by On-bead Digestion: On-bead enzyme digestion after immunoaffinity capture of target proteins has been widely used as an efficient approach for quantitative analysis of therapeutic proteins.20 As shown in Figure 4, instead of in solution method used in the workflow 1, an on-bead tagging reaction was performed in the current method, follow by removal of excess quenched TMPP reagent without elution of tagged protein from the magnetic beads. After the on-bead tryptic digestion of the tagged protein, digested peptide samples were cleaned up by SPE method prior to LC-MS/MS analysis. However, the 16 ACS Paragon Plus Environment

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sequence coverage of the target protein was lower than the results from the in-gel digestion method (Table 3), suggesting that on-bead digestion of NTs-huFc fusion protein may not be an efficient alternative to the in-gel digestion solution as tightly bound proteins on the magnetic beads may limit ready access of trypsin during the digestion process. Applying workflow 2 to the NTs-huFc protein both untagged N-terminus peptide and tagged N-terminus peptides were identified. While the detection of the untagged N-terminus was observed using workflow 2, compared to workflow 1, higher tagging efficiency was observed by this workflow. However, the incomplete on-bead tryptic digestion led to low ion intensity of the tagged N-terminus peptide and sub-optimal MS/MS data quality. Further, SPE based sample clean up may also have contributed to the lower sample recovery, as well as the potential for disproportionate loss of some hydrophilic peptides. Workflow 3: On-bead Tagging Followed by In-Solution Digestion. Building from successful application of the on-bead TMPP tagging step, the current workflow consists of the same onbead tagging process, but followed by elution of the tagged NTs-huFc fusion protein, and subsequent in-solution tryptic digestion. As shown in Table 3, 75% sequence coverage was observed from the enriched tagged protein sample. The N-terminus peptide identified with this workflow has an Xcorr of 2.36 which is much higher confidence level than workflow 2 with Xcorr of 0.69. Furthermore, no untagged N-terminus peptide was detected in this workflow which suggested excellent tagging efficiency was achieved. The high degree of labeling produced significantly higher ion intensity for the tagged N-terminus peptide, as well as improved MS/MS spectrum (Figure 5a, 5b). Combination of the on-bead tagging and in-solution tryptic digestion methods led to efficient selective TMPP tagging and minimal sample loss during sample preparation process. To evaluate the tagging consistency using workflow 3, 17 ACS Paragon Plus Environment


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triplicate runs were performed. In all runs, greater than 75% sequence coverage was achieved with 100% TMPP N-terminus tagging efficiency. Overall, workflow 3 produced the most consistent and highest quality data of the workflows tested. Application of the Workflow 3 to Study In Vitro Catabolic Stability of NTs-huFc Fusion Protein in Mouse Serum. In the in vitro mouse serum catabolic stability study, an aliquot of sample was taken at 0, 30, 60, and 120 min. All of the incubation samples were prepared using workflow 3 in preparation for LC-MS/MS analysis. Again, greater than 75% sequence coverage was achieved for all samples. As shown in Table 4, in addition to the N-terminus peptide of the NTs-huFc fusion protein, as well as CLP1 and CLP2, two additional TMPP tagged peptides were identified in the in vitro incubation samples. Even though both CLP1 and CLP2 were detected in the stock solution of the protein in low abundance, the significantly increased ion intensity of both tagged peptides in the incubation samples suggested additional proteolytic degradation of the protein at these two sites. The combined results suggest CLP1 and CLP2 are highly labile regions of the fusion protein and engineering efforts to reduce liability at these sites would be beneficial. In addition, identification of CLP2a in the incubation samples, but not in the stock solution indicated the in vitro degradation of the protein at the cysteine position. The identification of the fourth TMPP tagged peptide (CLP3) in the in vitro incubation samples led to the identification of another proteolytic degradation site at the valine of the bioactive portion of the fusion protein. Full MS scan peak areas of the TMPP tagged N-terminus peptide and CLP4 were plotted over the course of 120 min incubation (Figure 6). The plot revealed the disappearance of the intact NTs-huFc fusion protein with concomitant increase in CLP4 revealed


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synchronized formation of catabolite at the bioactive portion of the fusion protein. These results suggested potential simultaneous kinetic analysis of catabolic stability of the therapeutic protein and identification of its catabolites in the same experiment.



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Conclusions The work herein successfully demonstrated the combination of immunoaffinity capture of target proteins from complex biomatrices with a novel on-bead TMPP N-terminus tagging method to the study the catabolism of therapeutic proteins. Selective tagging of the N-terminus of the protein of interest with a hydrophobic positively charged TMPP, followed by LC-MS/MS analysis of the tryptic peptides allowed for fast and definitive identification of all N-terminus residues related to specific immuno-isolated proteins. The appearance of multiple N-terminal peptides can be related back to degradations of the primary therapeutic protein. This methodology as demonstrated leads to the unambiguous pinpoint of the proteolytic degradation sites in protein preparations as well as from in vivo samples. The optimized workflow was developed to focus on speed for routine catabolic stability information in drug discovery setting. A key step to improving the speed and efficiency of this workflow was due to the implementation of a novel on-bead TMPP tagging method that not only allowed highly selective and efficient tagging process, but also facilitated easy and complete removal of excess quenched TMPP reagent that precludes downstream LC-MS/MS analysis. The inherited high sensitivity of the peptide level LC-MS analysis provided opportunity to study in vitro and in vivo catabolism of therapeutic proteins at pharmacologically relevant, generally low concentrations relative to other protein in biomatrices such as serum. As a powerful orthogonal technique to the commonly used intact LC-MS method, this workflow can be readily used to study all possible Nterminus proteolytic degradations for a protein of interest.


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Acknowledgment The authors thank Dhanashri Bagal for review of this manuscript and for productive discussions of the experimental designs. The NTs-huFc fusion protein was kindly provided by Dr. Josh Pearson.



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REFERENCES 1. Reichert, J. M., Monoclonal antibodies as innovative therapeutics. Current pharmaceutical biotechnology 2008, 9 (6), 423-30. 2. Fishburn, C. S., The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. Journal of pharmaceutical sciences 2008, 97 (10), 4167-83. 3. Beck, A.; Reichert, J. M., Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. mAbs 2011, 3 (5), 415-6. 4. Hall, M. P., Biotransformation and in vivo stability of protein biotherapeutics: impact on candidate selection and pharmacokinetic profiling. Drug metabolism and disposition: the biological fate of chemicals 2014, 42 (11), 1873-80. 5. Hamuro, L. L.; Kishnani, N. S., Metabolism of biologics: biotherapeutic proteins. Bioanalysis 2012, 4 (2), 189-95. 6. Katsila, T.; Siskos, A. P.; Tamvakopoulos, C., Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry. Mass spectrometry reviews 2012, 31 (1), 110-33. 7. Vugmeyster, Y.; Harrold, J.; Xu, X., Absorption, distribution, metabolism, and excretion (ADME) studies of biotherapeutics for autoimmune and inflammatory conditions. The AAPS journal 2012, 14 (4), 714-27. 8. Stevenson, L.; Garofolo, F.; DeSilva, B.; Dumont, I.; Martinez, S.; Rocci, M.; Amaravadi, L.; Brudny-Kloeppel, M.; Musuku, A.; Booth, B.; Dicaire, C.; Wright, L.; Mayrand-Provencher, L.; Losauro, M.; Gouty, D.; Arnold, M.; Bansal, S.; Dudal, S.; Dufield, D.; Duggan, J.; Evans, C.; Fluhler, E.; Fraser, S.; Gorovits, B.; Haidar, S.; Hayes, R.; Ho, S.; Houghton, R.; Islam, R.; Jenkins, R.; Katori, N.; Kaur, S.; Kelley, M.; Knutsson, M.; Lee, J.; Liu, H.; Lowes, S.; Ma, M.; Mikulskis, A.; Myler, H.; Nicholson, B.; Olah, T.; Ormsby, E.; Patel, S.; Pucci, V.; Ray, C.; Schultz, G.; Shih, J.; Shoup, R.; Simon, C.; Song, A.; Neto, J. T.; Theobald, V.; Thway, T.; Wakelin-Smith, J.; Wang, J.; Wang, L.; Welink, J.; Whale, E.; Woolf, E.; Xu, R., 2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS. Bioanalysis 2013, 5 (23), 2903-18. 9. Ezan, E.; Becher, F.; Fenaille, F., Assessment of the metabolism of therapeutic proteins and antibodies. Expert opinion on drug metabolism & toxicology 2014, 10 (8), 1079-91. 10. Pearson, J. T.; Rock, D. A., Bioanalytical approaches to assess the proteolytic stability of therapeutic fusion proteins. Bioanalysis 2015, 7 (23), 3035-51. 11. Hager, T.; Spahr, C.; Xu, J.; Salimi-Moosavi, H.; Hall, M., Differential enzyme-linked immunosorbent assay and ligand-binding mass spectrometry for analysis of biotransformation of protein therapeutics: application to various FGF21 modalities. Analytical chemistry 2013, 85 (5), 2731-8. 12. Bults, P.; Bischoff, R.; Bakker, H.; Gietema, J. A.; van de Merbel, N. C., LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma. Analytical chemistry 2016, 88 (3), 1871-7. 13. Bertaccini, D.; Vaca, S.; Carapito, C.; Arsene-Ploetze, F.; Van Dorsselaer, A.; Schaeffer-Reiss, C., An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP. Journal of proteome research 2013, 12 (6), 3063-70. 14. Bland, C.; Hartmann, E. M.; Christie-Oleza, J. A.; Fernandez, B.; Armengaud, J., N-Terminaloriented proteogenomics of the marine bacterium roseobacter denitrificans Och114 using NSuccinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and diagonal chromatography. Molecular & cellular proteomics : MCP 2014, 13 (5), 1369-81. 15. Deng, J.; Zhang, G.; Huang, F. K.; Neubert, T. A., Identification of protein N-termini using TMPP or dimethyl labeling and mass spectrometry. Methods in molecular biology 2015, 1295, 249-58. 16. Chen, W.; Lee, P. J.; Shion, H.; Ellor, N.; Gebler, J. C., Improving de novo sequencing of peptides using a charged tag and C-terminal digestion. Analytical chemistry 2007, 79 (4), 1583-90. 17. Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M., In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols 2006, 1 (6), 2856-60. 22 ACS Paragon Plus Environment

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18. Gundry, R. L.; White, M. Y.; Murray, C. I.; Kane, L. A.; Fu, Q.; Stanley, B. A.; Van Eyk, J. E., Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 2009, Chapter 10, Unit10 25. 19. Gallien, S.; Perrodou, E.; Carapito, C.; Deshayes, C.; Reyrat, J. M.; Van Dorsselaer, A.; Poch, O.; Schaeffer, C.; Lecompte, O., Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol. Genome research 2009, 19 (1), 128-35. 20. Ackermann, B. L.; Berna, M. J., Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers. Expert review of proteomics 2007, 4 (2), 175-86.



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Tables Table 1. TMPP N-terminus tagged peptides in the control sample Name

Peptide Sequence

Charge (z)

m/z

N-terminus Peptide

QLYENKPR

3

534.96

CLP1

AKGQPR

3

410.19

CLP2

CKVSNK

2

645.77

Table 2. Impact of pH conditions to TMPP N-terminus tagging reaction pH

TMPP-NTT Peptide

Non-selective

Tagging efficiency (%)

TMPP-NTT (R and K)

6.4

0

0

-

7.4

0

0

-

8.2

3

1

34

Table 3. Summary of TMPP- N Terminus Workflows Workflow

Sequence

Untagged

Peptide Score

Tagging

Coverage

N-terminus

(Xcorr)

Efficiency

%

Peptide

Workflow 1

74

Detected

-

Incomplete

Workflow 2

71

Detected

0.69

Incomplete


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Workflow 3

75

Not Detected

2.36

Complete

Table 4. TMPP N-terminus tagged peptides identified in the in vitro mouse serum sample Peptide

TMPP-NTT

Name

Peptide

Time Points

0 N-terminus

QLYENKPR

c

++++

30

60

120

++++

+++

++

a

CLP1

AKGQPR

ND

+

++

+

a

CLP2

CKVSNK

++

++

++

++

CLP2a

CKVSNKALPAPIEK

++

+

+

+

VSNKALPAPIEK

+

+

++

++

b

b

CLP4

a

peptides identified in the control sample, b peptides identified only in the in vitro samples,

c

peak area measurements for each peptide at each time point, ranked from the highest (++++) to

the lowest (+), ND not detected.



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Figure Legend Figure 1. NTs-huFc Protein Sequence: Neurotensin peptide (italics), G4S linker (red), and human FC (bold). Figure 2. Identified TMPP Tagged Peptides. A) Extracted ion chromatogram of untagged Nterminus peptide (515.7697), clip 1 (410.1931), clip 2 (436.5238), and tagged N-terminus peptide (534.9095). B) MS/MS fragmentation of the untagged N-terminus peptide. C) MS/MS fragmentation of the tagged N-terminus peptide. D) MS/MS fragmentation of clip 1 peptide. E) MS/MS fragmentation of clip 2 peptide. Figure 3. Non-Selective TMPP Tagging at pH 8.2 of RPYILGGGGSDK. A) Extracted ion chromatogram of the untagged peptide (407.22014), and TMPP tagged peptide (448.7123). B) MS/MS fragmentation of the untagged RPYILGGGGSDK. C) MS/MS fragmentation of tagged RPYILGGGGSDK peptide. Figure 4. On-Bead TMPP-NTT Workflows Figure 5. On-Bead Tagging Followed By In-Solution Digestion. a) Extracted ion chromatogram of untagged N-terminus peptide (515.7698) and tagged N-terminus peptide (534.9096). b) MS/MS fragmentation of N-terminus tagged peptide using workflow 3. Figure 6. Peak Area Measurements of TMPP Tagged Peptide over Time. A) Peak area measurement of N-terminus peptide (QLYENKPR) over 4 time points (0, 30, 60, and 120 min). B) Peak area measurement of N-terminus peptide (VSNKALPAPIEK) over 4 time points (0, 30, 60, and 120 min).


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In Vitro

Trypsin digestion of tagged targeted protein

or

In Vivo

Isolate

Remove TMPP tagging of excess TMPP the targeted protein

Elution of tagged targeted protein

Peptide mapping analysis by LC-MS/MS

100 90 80

CLP1

70 60 50 40

CLP2 CLP1

Biofluid matrix

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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30 20 10

CLP2

0

22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

for TOC only “image courtesy of Majlinda Kullolli”, Copyright 2016 ACS Paragon Plus Environment

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Figure 1.

QLYENKPRRPYILGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

ACS Paragon Plus Environment

100 90 80 70 60 50 40 30 20 10 0

N-Terminus Untagged Peptide (Peak 1) RT: 20.82 MH: 4.1E5 BP: 515.7697

CLP1 Tagged Peptide (Peak 3) RT: 38.03 CLP2 MH: 1.6E5 BP: 410.1905 Tagged Peptide (Peak 2) RT: 37.68 MH: 8.0E4 BP: 436.5258

21.02

20

25

30

Relative Abundance

ms2 [email protected] [136.0000-1042.0000] NL: 6.67E2 [M+2H]2+ -NH3–H2O 498 100 90 80 y5+ 70 y2+ 60 272 50 40 y62+ 404 30 + 2+ b y 7 4 20 258 857 10 0 200 300 400 500 600 700 800 900 m/z

D

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35 Time (min)

40

N-Terminus Tagged Peptide (Peak 4) RT: 46.37 MH: 4.5E4 BP: 534.9095 45

50

C ms2 [email protected] [142.0000-1615.9999] NL: 1.77E3 [M+3H]3+ -H2O, [M+3H]3+ -NH3 y42+529 100 514 90 80 y2+ 272 70 + b y72+ + 60 4 b3 1089 460 960 50 + y 3 + 40 y1 400 175 30 b5 + + 20 1203 b2+63+ y5 b72+ 2+ y b3 6 10 0 200 1000 400 600 800 1000 1200 1400 m/z Relative Abundance

A

B

E

Relative Abundance

ms2 [email protected] [115.0000-1318.9999] NL: 6.72E3 ms2 [email protected] [107.0000-1241.0000] NL: 4.96E3 + + y y2 4 447 272 100 100 3+ [M+3H] -NH3–H2O 90 90 398 80 y3+ 80 348 70 70 60 60 b42+ b32+ 479 50 50 b 2+ 480 772 40 40 2+ b22+ y4 - NH3 30 30 861 y42+ 229 224 20 20 10 10 0 0 200 400 600 800 1000 1200 200 300 400 500 600 700 800 900 1000 1100 1200 ACS Paragon Plus Environment m/z m/z

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Relative Abundance

Figure 2.


1600

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Figure 3.

A

RT: 25.98 MH: 1.6E5 BP: 407.2204

100

m/z= 407.2199407.2215+ 448.7115448.7133

90

Relative Abundance

80 70 60 50 40 RT: 48.52 MH: 2.5E4 BP: 448.7123

30 20 10 0 0

10

20

30

40 Time (min)

50

60

C ms2 [email protected] [107.0000-1230.9999] NL: 1.42E3 y7+ 577 2+ 3+ 100 b5 y10 322 90 + b 4 80 530 70 60 b 5+ 50 643 40 + y 5 30 y62+ 463 y8+ 20 260 690 10 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

ms2 [email protected] [118.0000-1804.0000] NL: 2.01E3 100 90 80 70 60 50 40 30 20 10 0

Relative Abundance

B

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b42+ 552 b53+,y4+b 3+,y + 8 5 b62+ 406 463 637 b72+ 666

y72+ 289

200


400

600

800

1000 1200 1400 1600 1800 m/z

Figure 4. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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1000 ng/mL spiked sample in mouse serum (200uL of sample) Pull Down using M280 crosslinked with Ab35 The crosslinked beads were incubated with the samples for 4 hrs at 4 ͦC

The beads were washed using KingFisher (1hr)

Workflow 1.

Workflow 2.

Workflow 3.

Protein elution

TMPP tagging/quenching on bead (2 hr)

TMPP tagging/quenching on bead

In-Solution TMPP tagging/quenching (2hr)

Washing the excess TMPP from the beads (45 min)

Washing the excess TMPP from the beads (45 min)

In-gel Digestion (2 days)

On-bead digestion (overnight)

Sample concentration (1hr)

Sample cleanup/concentration (2hrs) ACS Paragon Plus Environment

Protein elution

Sample concentration/trypsin digestion (overnight)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

RT: 47.60 MH: 2.24E6 BP: 534.9096

100

m/z= 515.7688-515.7708+ 534.9084-534.9106

90 80 70 60

50 40 30 20 10 0

B

0

10

20

30

40 Time (min)

50

60

ms2 [email protected] [142.0000-1614.0000] NL: 1.97E4 y2+ 272

100

[M+3H]3+ -H2O, [M+3H]3+ -NH3 529

90 80 Relative Abundance

Figure 5.

Relative Abundance

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b3+ 960

+

70

+

y3 400

60

y4 514

b4+ 1089

50 40 30

y1+ 175

20

b5+ 1203

10 0

200

400

600

800


m/z

1000

1200

1400

1600


Figure 6. A

B

VSNKALPAPIEK

QLYENKPR 5.0 ´ 10 7

6´ 10 6

4.0 ´ 10 7 3.0 ´ 10

P ea k A re a

Peak Area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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7

2.0 ´ 10 7 1.0 ´ 10 7

5´ 10 6

4´ 10 6

3´ 10 6

0 0

15 30 45 60 75 90 105 120 135

Time (min)

0

15 30 45 60 75 90 105 120 135

Time (min)


Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to

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