key: cord-0929145-72pjwh72 authors: Tripathi, Gaurav; Sharma, Nupur; Bindal, Vasundhra; Yadav, Manisha; Mathew, Babu; Sharma, Shvetank; Gupta, Ekta; Singh, Jaswinder; Sarin, Shiv Kumar title: Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis date: 2021-11-27 journal: STAR Protoc DOI: 10.1016/j.xpro.2021.101045 sha: e3aae5b5eb0a7c319c2a912d94088ff3625cccb2 doc_id: 929145 cord_uid: 72pjwh72 In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, Patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS) based analysis, and downstream bioinformatics analysis. Add 500 mL of HPLC-grade methanol to 300 mL of HPLC-grade water, Add 100 mL of reagent 129 grade acetic acid mix and adjust final volume to 1000 mL with water. The final concentrations 130 will be 50% (v/v) methanol in water with 10% (v/v) acetic acid. 131 (Note-By the end of this step, the excel data files would be curated). 307 (Note -The steps for data curation from raw files for Proteome, Metaproteome, and virome are 308 the same, and the same raw files can be used for data curation analysis of all three. 309 The only difference among the three is that each need to be run individually against their 310 respective database, e.g., Host proteome should be mapped against Human Uniprot database) 311 86. Open Proteome discoverer 2.2. 312 87. Click New Study. (Figure 2 ) 313 88. Name your study > Decide the root directory > Add your raw files > Press ok. (Figure 3 ) 314 89. Click on New Analysis. (Figure 4 ) 315 90. Click on Input files > Select all files and drag it below the processing workflow window > Click 316 on the icon of the processing step. (Figure 5 ) 317 91. Click on Open common > Select your processing workflow and press enter. (Figure 6 ) 318 COVID virome, select the already curated COVID database> Select the type of digestion we 320 performed, e.g., tryptic peptides. (Figure 7 ) 321 (Note-This is the place where you will select the database for proteome, metaproteome, and 322 virome or any other organism associated) 323 93. Now, select each step of the Processing workflow. 324 (Note-Setting for each parameter group is given in the image below. (Figure 8 ) 325 94. Now click on consensus workflow > then Open common > and select your workflow. (Figure 326 9) Step 10-Now select each step of the processing workflow. 327 (Note-Setting for each parameter group is given in the image below. (Figure 10 ) 328 95. Click on grouping and quantification> Then select Sample type. (Figure 11 ) 329 96. Click on Run. (Figure 12 Visit the Unipept website https://unipept.ugent.be/. (Figure 15 ) 347 Enter the list of tryptic peptides here > Click search (Figure 16) . 348 103. You will get a sunburst plot from Unipept ( Figure 17) . 349 104. Now download the data from Unipept by clicking on the download tab. (Fig-18) . 350 You will get a file like this (Figure 19) . 351 Repeat these steps for the other two groups. 352 Now, you will have this excel file for all groups. 353 Now, compile all three files and master peptide files to get the abundance of each bacterium 354 (Figure-20) . 355 During this process, you can identify each disease group-specific metaproteome and 356 compile the relative abundance of metaproteome. protocol describes an approach that combines the Sample collection, virus inactivation, protein 397 isolation, alkylation, digestion, desalination, and peptide sequencing using a Q-exactive + mass 398 spectrometer bioinformatics approaches for data analysis like database curation, using proteome 399 discoverer 2.2 and metaproteome analysis. If still the problem persists, reprepare your samples, and this time before using any plastic ware, 429 soak them in any organic solvent (e.g., ethanol or acetone) for 24-72 hours to remove any 430 contaminant polymer. 431 The trap column of Nano-LC gets blocked. (Step 83-87) . 433 In such a scenario, first, you disconnect the column from source > then reverse the direction of 435 The air bubble got trapped in the nano-LC pump. (Step 83-87) . 440 In such a scenario, first, you disconnect the column from source > then join the column end with 442 waste outlet> then start purge > the flush finally performs a leak test if pass. Rearrange the 443 column and start. 444 Problem 4: (Step 83-87) . 445 In such a scenario, check that the lamp is on and the cables are well connected. Next, ensure 447 that the flow is normal and the automatic sampler is working properly, not blocked or 448 jammed. 449 Problem 5: 450 No or low protein detection- (Step 60-82) . The study did not generate any materials. 465 No data is provided, and the raw data for the manuscript is available on request to the lead 467 contact Dr. Jaswinder Singh Maras (jassi2param@gmail.com) . There is no conflict of interest from any of the authors included in the manuscript. 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