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JPR Submission This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Journal of Proteome Research, copyright ©American Chemical Society after peer review. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acs.jproteome.9b00487. Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform. Please provide feedback Please support the ScholarWorks@UMBC repository by emailing [email protected] and telling us what having access to this work means to you and why it’s important to you. Thank you. Journal of Proteome Research This document is confidential and is proprietary to the American Chemical Society and its authors. Do not copy or disclose without written permission. If you have received this item in error, notify the sender and delete all copies. Top-down Proteomic Characterization of Truncated Proteoforms Journal: Journal of Proteome Research Manuscript ID pr-2019-004879.R1 Manuscript Type: Article Date Submitted by the 10-Sep-2019 Author: Complete List of Authors: Chen, Dapeng; University of Maryland at College Park; Zeteo Tech Inc Geis-Asteggiante, Lucia; University of Maryland at College Park Gomes, Fabio; Scripps Research Institute, Department of Molecular Medicine Ostrand-Rosenberg, Suzanne; University of Maryland Baltimore County Fenselau, Catherine; University of Maryland at College Park ACS Paragon Plus Environment Page 1 of 14 Journal of Proteome Research 1 1 2 3 4 Top-down Proteomic Characterization of Truncated Proteoforms 5 6 7 1,3 1,4 1,5 2 8 Dapeng Chen , Lucia Geis-Asteggiante , Fabio P Gomes , Suzanne Ostrand-Rosenberg , Catherine 9 Fenselau1 * 10 11 1Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, United 12 States; 2Department of Biological Sciences, University of Maryland Baltimore County, Baltimore MD 13 21250, United states; 3Current address Zeteo Tech Inc, Sykesville MD 21784, United States; 4Current 14 address University Hospital Frankfurt, Frankfurt, Germany; 5Current address The Scripps Research 15 16 Institute, La Jolla CA 92037, United States. 17 *To whom correspondence should be addressed: [email protected] 18 19 20 21 ABSTRACT: A top-down proteomic strategy with semi-automated analysis of data sets has proven 22 successful for global identification of truncated proteins without the use of chemical derivatization, 23 24 enzymatic manipulation, immunoprecipitation or other enrichment. This approach provides the reliable 25 identification of internal polypeptides formed from precursor gene products by proteolytic cleavage of 26 both the N- and C-termini, as well as truncated proteoforms that retain one or the other termini. The 27 strategy has been evaluated by application to the immunosuppressive extracellular vesicles released by 28 29 myeloid-derived suppressor cells. More than 1000 truncated proteoforms have been identified, from 30 which binding motifs are derived to allow characterization of the putative proteases responsible for 31 truncation. 32 33 KEYWORDS: truncated proteoforms, top-down mass spectrometry, cleavage motifs, protease database, 34 extracellular vesicles, myeloid-derived suppressor cells 35 36 37 38 INTRODUCTION 39 40 Truncation of proteins gives rise to new proteoforms that have the potential to play important 41 roles in regulating signal transduction and cell activity.1-5 Several mechanisms can generate truncated 42 proteins, including degradation of full-length proteins and, more rarely, alternative start and stop sites 43 for transcription or protein translation. Studies of truncated proteoforms have been challenging 44 45 because the intracellular abundances of most protease products are low, and selective enrichment has 46 been required prior to mass spectrometric or other characterization.4, 6-8 Thus far, most analyses on the 47 proteomic scale have depended on bottom-up strategies with tryptic cleavage, and have targeted 48 polypeptides with new derivatized amino-termini for identification or quantitation.1-3, 6-9 In the present 49 50 study top-down mass spectrometry is extended using semiautomated bioinformatic processing, and 51 demonstrated to identify and characterize truncated polypeptides without chemical or enzymatic 52 manipulation, immunoprecipitation or other selective enrichment. We identify polypeptide sequences 53 released from both sides of scissile bonds in precursor proteins, and for the first time characterize 54 internal products formed by multiple cleavages in a reliable high throughput strategy. Our approach is 55 56 evaluated by analysis of the lysate of extracellular vesicles shed by cancer-induced myeloid-derived 57 58 59 60 ACS Paragon Plus Environment Journal of Proteome Research Page 2 of 14 2 1 2 3 suppressor cells (MDSC). This sample was chosen for several reasons: (i) it is present in virtually all 4 cancer patients and a major obstacle to antitumor immunity;10-13 (ii) we have previously identified many 5 6 of the proteins carried by these extracellular vesicles, and demonstrated that some proteins and 7 ubiquitinated proteoforms mediate immune suppressive functions of intact MDSC;11, 14,15 and (iii) 8 because truncated proteins have been detected frequently in MDSC-derived extracellular vesicles.10,11 9 Truncated proteins have also been observed in urinary extracellular vesicles.16 One source of truncated 10 11 proteins in extracellular vesicles might be polypeptides formed in parental cells and selectively removed 17-21 12 by extracellular vesicles. An alternative or co-existing explanation is that proteins are hydrolyzed in 13 situ by proteases active in the extracellular vesicles. This latter hypothesis is consistent with the previous 14 identification of 49 proteases and the 37 components of the proteasome and immuno-proteasome in 15 MDSC-derived extracellular vesicles11,22 and of 34 proteases in urinary extracellular vesicles.23 Protease, 16 17 proteasome and other enzymatic activities have recently been demonstrated to occur in situ in several 18 kinds of extracellular vesicles.23-28 Whether proteolysis occurs in one or both locations, truncated 19 proteoforms in MDSC-derived extracellular vesicles provide novel polypeptides potentially able to 20 impact and modulate the significant biological functions of extracellular vesicles, e.g., intercellular 21 communication, regulation and dysregulation, immunomodulation and therapeutics. 22 23 Our first objective was to establish a successful top-down strategy to identify truncated 24 proteoforms. Subsequent objectives included characterization of the variety of truncated polypeptides 25 26 present in these biologically active extracellular vesicles, examination of cleavage sites in the precursor 27 proteins, and consideration of the cellular and extracellular proteases responsible for the observed 28 truncations. 29 30 31 32 33 EXPERIMENTAL SECTION 34 35 Myeloid-derived suppressor cells were obtained from mice carrying syngeneic primary and metastatic 36 4T1 mammary tumors and allowed to shed extracellular vesicles as previously reported.11, 14 Extracellular 37 38 vesicles were lysed by suspension in 8 M urea in 50 mM ammonium bicarbonate (pH 8.5). A protease 39 inhibitor cocktail (1% by volume) (Sigma Aldrich, St. Louis MO) and a deubiquitinase inhibitor PR619 (50 40 µM) (LifeSensors, Malvern PA) were included to control artifactual proteolysis. Samples were buffer 41 exchanged with 50 mM ammonium bicarbonate (pH 8.5) taking final samples to 0.8 M urea. Protein 42 content was measured using the Pierce BCA Assay Kit (Thermo Scientific, San Jose CA). Seven mouse 43 samples were pooled to provide approximately 600 µg protein for an analysis that included fractionation 44 by gel electrophoresis in solution. An additional biological replicate was analyzed using material from a 45 single mouse experiment, approximately 100 µg protein. 46 o 47 Proteins in the pooled preparation were precipitated at -20 C for 1 hr, resolubilized in 1% SDS, 48 and fractionated using gradient electrophoresis in solution following the manufacturer’s instructions 49 (Expedeon GELFrEE, San Diego CA). Twelve fractions were collected and evaluated by 1D gel 50 electrophoresis. Fractions were pooled 1-2, 3-8, 9-10 and 11-12 according to similarities in protein sizes 51 as visualized by 1-D gels (See Supporting Figure S-1), precipitated from the SDS solution with 52 53 CHCl3:CH3OH:H2O (4:1:3), and resolubilized in H2O:ACN (97.5:2.5) with 0.1% formic acid (solvent A). 54 Each pooled fraction was desalted and concentrated in a PepSwift RP-4H monolith trap (100 µm x 5 mm) 55 at a flow rate of 5 μL/min, and separated on a ProSwift RP-4H monolith column 200 µm x 25 cm 56 57 58 59 60 ACS Paragon Plus Environment Page 3 of 14 Journal of Proteome Research 3 1 2 3 (Thermo Fisher) using a gradient of 5 to 55% solvent B ACN:H2O (75:25) 0.1% formic acid at a flow rate 4 of 1.0 µL/min through 120 min. The biological replicate of smaller sample size was not fractionated. It 5 6 was desalted and concentrated on a PepSwift RP-4H monolith trap into 5% solvent B and subsequently 7 separated using a ProSwift RP-4H monolith column. A gradient of 5 to 55% solvent B was developed 8 through 145 min. Five replicate injections were made. 9 10 Top-down LC-MS/MS analysis was carried out using an Ultimate 3000 RSLCnano system (Dionex, 11 Sunnyvale CA) coupled via a Nanospray Flex (Thermo Fisher Scientific) to a Fusion Lumos tribrid orbitrap 12 mass spectrometer (Thermo Fisher Scientific).29,30 Resolving power of the orbitrap was set to 120,000 at 13 14 m/z 200 for both precursor and product ion analysis. Precursor spectra were acquired in intact protein 15 mode. MS1 and MS2 spectra were acquired by averaging 2 to 5 microscans. MS/MS spectra were 16 produced in “top-speed” data-dependent mode with a fixed duty cycle of 10s and dynamic exclusion of 17 60 sec. Precursor ions with a charge state of +8 and higher were selected for activation.
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