Wang et al. Clin Proteom (2021) 18:16 https://doi.org/10.1186/s12014-021-09322-0 Clinical Proteomics RESEARCH Open Access Quantitative proteomic analysis of aberrant expressed lysine acetylation in gastrointestinal stromal tumors Bo Wang1,2†, Long Zhao1,3†, Zhidong Gao1, Jianyuan Luo4, Haoran Zhang1, Lin Gan1, Kewei Jiang1, Shan Wang2,3, Yingjiang Ye1 and Zhanlong Shen2,3* Abstract Background: Gastrointestinal stromal tumor (GIST) is a common digestive tract tumor with high rate of metastasis and recurrence. Currently, we understand the genome, transcriptome and proteome in GIST. However, posttranscrip- tional modifcation features in GIST remain unclear. In the present study, we aimed to construct a complete profle of acetylome in GIST. Methods: Five common protein modifcations, including acetylation, succinylation, crotonylation, 2-hydroxyisobu- tyrylation, and malonylation were tested among GIST subgroups and signifcantly diferentially- expressed lysine acetylation was found. The acetylated peptides labeled with Tandem Mass Tag (TMT)under high sensitive mass spec- trometry, and some proteins with acetylation sites were identifed. Subsequently, these proteins and peptides were classifed into high/moderate (H/M) risk and low (L) risk groups according to the modifed NIH classifcation standard. Furthermore, cell components, molecular function, biological processes, KEGG pathways and protein interaction networks were analyzed. Results: A total of 2904 acetylation sites from 1319 proteins were identifed, of which quantitative information of 2548 sites from 1169 proteins was obtained. Finally, the diferentially-expressed lysine acetylation sites were assessed and we found that 42 acetylated sites of 38 proteins were upregulated in the H/M risk group compared with the L risk group, while 48 acetylated sites of 44 proteins were downregulated, of which Ki67 K1063Ac and FCHSD2 K24Ac were the two acetylated proteins that were most changed. Conclusions: Our novel fndings provide further understanding of acetylome in GIST and might demonstrate the possibility in the acetylation targeted diagnosis and therapy of GIST. Keywords: Gastrointestinal stromal tumor, Acetylation, Proteomics, PTM Background GISTS were originally known as leiomyomas or epithe- Gastrointestinal stromal tumors (GISTs) are the most lial leiomyosarcoma because GISTs mostly occur in the common type of mesenchymal tumors of the gastrointes- muscular layer of hollow organs [1, 2]. Until 1983, Mazur tinal tract and originate from interstitial cells of Cajal [1]. and Clark [3] used the name gastrointestinal stromal tumors, including all interstitial-derived tumors, as well as non-epithelial tumors of varying degrees of diferentia- *Correspondence: [email protected] †Bo Wang and Long Zhao contributed equally to this work tion, such as leiomyoma and Schwannoma. GISTs mostly 2 Laboratory of Surgical Oncology, Peking University People’s Hospital, occur in the elderly, usually under the mucosa, but also in Beijing, People’s Republic of China other parts of the gastrointestinal tract and are common Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Clin Proteom (2021) 18:16 Page 2 of 15 in the stomach [4]. Deepening our understanding of the Research Ethics Committee of Peking University People’s research has led to further understanding of the pathol- Hospital (Beijing, China). ogy and treatment of this disease. At present, most GISTs have shown to bear c-kit proto-oncogene mutations, and Protein extraction express CD117 and CD34 [5, 6]. KIT mutations are most Samples were taken out of the − 80 °C, and about 1 g tis- common, and using molecular analysis, several mutation sue sample was weighed into a mortar pre-cooled with locations have been identifed, including exons 9, 11, 13, liquid nitrogen, then grinded in liquid nitrogen into cell and 17 [7]. Up to now, there is no authoritative and accu- powder, and transferred to a 5-mL centrifuge tube. Next, rate epidemiological data on GISTs in China. Relevant four volumes of lysis bufer (8 M urea, 1% Protease Inhib- medical research in the United States of America shows itor Cocktail) was added to the cell powder, followed by that GISTs have an annual incidence of 11.0 to 19.6 indi- three sonications on ice using a high intensity ultrasonic viduals/1 million individuals [8, 9]. processor (Scientz, Ningbo, China). For PTM experi- Imatinib mesylate, a small molecule inhibitor of KIT ments, inhibitors were added to the lysis bufer, including and PDGFRA, yields long-lasting responses in most 3 μM TSA (Trichostatin A) and 50 mM NAM (Nicotina- patients, with a median survival of almost 5 years [10]. mide) for acetylation. Te remaining debris was removed However, 20% of patients show primary resistance by centrifugation at 12,000 g at 4 °C for 10 min. Finally, to imatinib, and most responding patients eventually the supernatant was collected and the protein concen- develop secondary resistance and disease progression tration was determined using BCA kit according to the [11, 12]. Te response rate for second-line treatment with manufacturer’s instructions. sunitinib was 7%, however, patients progressed after an average of 6 months, and the results at the time of pro- Western blot assay gression were frustrating [13]. Consequently, there is an Total proteins were extracted from 9 GISTs tissue sam- unmet need for alternative treatment strategies to treat ples (3 of each grade) lysis bufer. Lysates were denatured GISTs. with SDS sample bufer at 95 °C for 5 min, and proteins Evidence has shown that protein post-translational were separated in 8–12% polyacrylamide gels, then trans- modifcation (PTM) might contribute as a complement ferred to nitrocellulose blotting (NC) membranes (Mil- of therapeutic target for malignant tumors [14, 15]. Ini- lipore, Massachusetts, United States). Membranes were tially and during progression, epigenetic alteration, blocked with 5% non-fat milk powder in TBST bufer which leads to modifed gene expression was involved for an hour at home temperature, and incubated with in GISTs, [16–18]. Te type of PTM that plays a central primary antibodies overnight at 4 °C. Anti-acetyllysine, role in GISTs is still unknown. Moreover, no informa- anti-succinyllysine, anti-crotonyllysine, anti-2-hydroxy- tion is available about the target of protein PTM in the isobutyryllysine, and anti-malonyllysine antibodies were treatment of GISTs. Terefore, it is necessary to identify used as the primary antibodies. All of the primary anti- the main PTM and the diferentially-expressed PTM pro- bodies were purchased from PTM Biolabs lnc. (Hang- teins. In this study, we discovered lysine acetylation as zhou, China). Ten, membranes were washed with TBST the major changed PTM in GISTs, and used integrated bufer and incubated with the second antibody Goat anti- approach involving tandem mass tag (TMT) labeling and Mouse IgG (Pierce™, Termo Scientifc, USA) at room mass spectrometry-based quantitative proteomics to temperature for 45 min. quantify dynamic changes of protein acetylation includ- ing histone or non-histone proteins among GISTs sam- Trypsin digestion ples of diferent grades. For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C, and alkylated Methods with 11 mM iodoacetamide for 15 min at room temper- Human tissue specimens ature in the dark. Ten, the protein sample was diluted After the patient was pathologically diagnosed with by adding 100 mM TEAB to a urea concentration of GIST, human tissue specimens were collected periopera- less than 2 M. Finally, for the frst digestion, trypsin was tively. Tissue samples were obtained in less than 30 min added at a 1:50 trypsin-to-protein mass ratio overnight of ischemia time, then quickly frozen in liquid nitrogen and 1:100 trypsin-to-protein mass ratio was added for a until use. At each risk level (high risk, moderate risk and second 4 h-digestion. low risk groups), we selected three patients’ clinical tissue samples for subsequent mass spectrometry testing. Writ- TMT labeling ten informed consent was provided by all patients before After trypsin digestion, peptides were desalted by a Strata sample collection. Tis study was approved by the local X C18 SPE column (Phenomenex, California, United Wang et al. Clin Proteom (2021) 18:16 Page 3 of 15 States) and vacuum-dried. Peptides were reconstituted in Trypsin/P was specifed as the cleavage enzyme allowing 0.5 M TEAB and processed according to the manufactur- up to 4 missing cleavages. Te mass tolerance for precur- er’s guidelines provided with the TMT kit. In brief, one sor ions was set to 20 ppm in the First search, 5 ppm in unit of TMT reagent was thawed and reconstituted in the Main search, and the mass tolerance for fragment acetonitrile. Subsequently, peptide mixtures were incu- ions was set to 0.02 Da. Carbamidomethyl on Cys was bated for 2 h at room temperature and pooled, desalted, specifed as fxed modifcation and acetylation modif- and dried by vacuum centrifugation.