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Depending on the different type of samples there are different mass spectrometry instrument that can be selected. 

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Differences among search engines can include the scoring algorithm, whether it considers data quality, and whether it considers all fragment ion types or only a subset. Often, the best way to select an algorithm is to examine the details regarding data-format compatibility and necessary features, test it on a data set, and compare the results to what is expected. 

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After the MS data is collected, the next step is to perform a database search to identify the proteins and/or peptides analyzed by MS. A number of parameters can affect the outcome of a database search, and, ultimately, search parameters should consider the history of the sample, such as organism, enzymatic or chemical digestion, and reduction/alkylation steps. 

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}, "position": 1 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2258, "name": "Protein preparation: Polyacrylamide gel", "due_date": null, "description": null, "x": 0, "y": 0, "my_module_group_id": 1179, "created_at": "2018-11-26T12:04:02.091Z", "updated_at": "2018-12-19T09:02:54.887Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 2, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5978, "input_id": 2345, "output_id": 2258 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3678, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2258, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-26T12:04:02.098Z", "updated_at": "2019-02-20T07:32:07.687Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5146, "name": "Perform enzymatic digestion", "position": 5, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T11:40:40.624Z", "updated_at": "2018-12-24T06:40:41.505Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 942, "name": "Guidelines:", "step_id": 5146, "created_at": "2018-12-20T14:15:25.669Z", "updated_at": "2018-12-20T14:15:25.669Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3869, "text": "Dilute the gel enzyme stock solution ~1:1000 with 25 mM ammonium bicarbonate to obtain a 10 to 20 μg/mL working solution.", "checked": false, "checklist_id": 942, "created_at": "2018-12-20T14:15:25.671Z", "updated_at": "2018-12-20T14:15:25.671Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3870, "text": "Add a sufficient amount of the gel enzyme working solution to cover gel pieces and incubate on ice for 1 hr. For a typical gel band/spot, this is ~10 to 20 μL.", "checked": false, "checklist_id": 942, "created_at": "2018-12-20T14:15:25.679Z", "updated_at": "2018-12-20T14:15:25.679Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3871, "text": "Remove excess gel enzyme solution, then add sufficient 25 mM NH4HCO3 to cover the gel pieces. This increases the pH and thereby inhibits the enzymatic digestion for those enzymes which have low pH optima.", "checked": false, "checklist_id": 942, "created_at": "2018-12-20T14:15:25.686Z", "updated_at": "2018-12-20T14:15:25.686Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3872, "text": "Incubate at 37°C overnight.", "checked": false, "checklist_id": 942, "created_at": "2018-12-20T14:15:25.693Z", "updated_at": "2018-12-20T14:15:25.693Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 } ] }, { "step": { "id": 5145, "name": "Reduce disulfide bonds and alkylate free cysteines", "position": 4, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T11:01:14.616Z", "updated_at": "2018-12-20T14:13:35.200Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nDithiothreitol (DTT) is a reducing agent that converts cystine’s disulfide bond into cysteine’s free sulfhydryl groups.
Iodoacetamide (IAA) is an alkylating agent that reacts with free sulfhydryl groups of cysteine residues to form S-carboxyamidomethyl-cysteine, which cannot be reoxidized to form disulfide bonds. This is important for allowing trypsin maximum access to cleavage sites within the protein.\n" }, "position": 0 }, { "checklist": { "checklist": { "id": 941, "name": "Guidelines:", "step_id": 5145, "created_at": "2018-12-20T14:12:18.059Z", "updated_at": "2018-12-20T14:12:18.059Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3865, "text": "Add 100 μL of 10 mM dithiothreitol to the gel piece, then incubate 45 min at 55°C.", "checked": false, "checklist_id": 941, "created_at": "2018-12-20T14:12:18.062Z", "updated_at": "2018-12-20T14:12:18.062Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3866, "text": "Remove solution, add 100 μl 55 mM iodoacetamide, and incubate 30 min at room temperature in the dark.", "checked": false, "checklist_id": 941, "created_at": "2018-12-20T14:12:18.070Z", "updated_at": "2018-12-20T14:12:18.070Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3867, "text": "Remove IAA solution and add 400 μL gel wash solution, incubate at room temperature, and shake for 15 min. Repeat this step twice.", "checked": false, "checklist_id": 941, "created_at": "2018-12-20T14:12:18.077Z", "updated_at": "2018-12-20T14:12:18.077Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3868, "text": "Dehydrate gel pieces with 400 μL of 100% acetonitrile for 10 min, then remove supernatant and dry gel pieces using a vacuum centrifuge.", "checked": false, "checklist_id": 941, "created_at": "2018-12-20T14:12:18.083Z", "updated_at": "2018-12-20T14:12:18.083Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 } ] }, { "step": { "id": 5147, "name": "Extract peptides", "position": 6, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T11:52:09.928Z", "updated_at": "2018-12-24T06:42:22.727Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Here is the guideline how to extract peptides. This step can take up to several hours depending upon the volume used.

" }, "position": 0 }, { "checklist": { "checklist": { "id": 943, "name": "Guidelines:", "step_id": 5147, "created_at": "2018-12-20T14:17:36.750Z", "updated_at": "2018-12-20T14:17:36.750Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3873, "text": "Remove supernatant, which now contains peptides, to a new clean microcentrifuge tube.", "checked": false, "checklist_id": 943, "created_at": "2018-12-20T14:17:36.754Z", "updated_at": "2018-12-20T14:17:36.754Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3874, "text": "Add 100 μL gel extraction solution to the gel pieces and incubate while shaking for 20 min at room temperature.", "checked": false, "checklist_id": 943, "created_at": "2018-12-20T14:17:36.761Z", "updated_at": "2018-12-20T14:17:36.761Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3875, "text": "Remove solution and combine with the solution obtained in step 1.", "checked": false, "checklist_id": 943, "created_at": "2018-12-20T14:17:36.777Z", "updated_at": "2018-12-20T14:17:36.777Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3876, "text": "Repeat steps second and third step.", "checked": false, "checklist_id": 943, "created_at": "2018-12-20T14:17:36.784Z", "updated_at": "2018-12-20T14:17:36.784Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3877, "text": "Dry combined supernatants using a vacuum centrifuge.", "checked": false, "checklist_id": 943, "created_at": "2018-12-20T14:17:36.791Z", "updated_at": "2018-12-20T14:17:36.791Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] }, "position": 1 } ] }, { "step": { "id": 4826, "name": "Coomassie stained gels", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-26T13:15:06.518Z", "updated_at": "2018-12-24T06:34:58.049Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nCoomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels. The gels are soaked in dye and an excess stain is then eluted with a solvent. This treatment allows the visualization of protein bands." }, "position": 0 }, { "checklist": { "checklist": { "id": 917, "name": "Washing:", "step_id": 4826, "created_at": "2018-12-07T10:52:29.542Z", "updated_at": "2018-12-07T11:39:18.108Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3780, "text": "Remove destain solution", "checked": false, "checklist_id": 917, "created_at": "2018-12-07T10:52:29.545Z", "updated_at": "2018-12-07T10:52:29.545Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3781, "text": "Add 400 μl water.", "checked": false, "checklist_id": 917, "created_at": "2018-12-07T10:52:29.554Z", "updated_at": "2018-12-07T10:52:29.554Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3782, "text": "If the gel pieces are still blue, remove the water and repeat until the gel pieces are colourless.", "checked": false, "checklist_id": 917, "created_at": "2018-12-07T10:52:29.562Z", "updated_at": "2018-12-07T10:52:29.562Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 }, { "checklist": { "checklist": { "id": 919, "name": "Dehydration:", "step_id": 4826, "created_at": "2018-12-07T11:39:18.116Z", "updated_at": "2018-12-07T11:39:18.116Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3786, "text": "Dehydrate gel pieces with 400 μl of 100% acetonitrile for 10 min", "checked": false, "checklist_id": 919, "created_at": "2018-12-07T11:39:18.118Z", "updated_at": "2018-12-07T11:39:18.118Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3787, "text": "Remove supernatant", "checked": false, "checklist_id": 919, "created_at": "2018-12-07T11:39:18.126Z", "updated_at": "2018-12-07T11:39:18.126Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3788, "text": "Dry gel pieces in a vacuum centrifuge", "checked": false, "checklist_id": 919, "created_at": "2018-12-07T11:39:18.133Z", "updated_at": "2018-12-07T11:39:18.133Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 2 } ] }, { "step": { "id": 4824, "name": "Destain gel bands", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-26T13:08:09.568Z", "updated_at": "2019-01-31T08:32:58.429Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Destain in the microcentrifuge tube for 30 min with 100 μL of the appropriate gel destain solution with vigorous vortexing. The choice of destaining protocol depends on the stain used, and there is a specific destain recipe for each type of gel stain. The choice of protein stain depends on the protein concentration and the dynamic range of the sample. Silver and colloidal Coomassie staining (CCS) stains offer a low limit of detection (1 to 10 ng) compared to traditional Coomassie staining (0.3 to 1 μg).

" }, "position": 0 }, { "checklist": { "checklist": { "id": 940, "name": "Follow:", "step_id": 4824, "created_at": "2018-12-20T14:03:52.360Z", "updated_at": "2018-12-20T14:03:52.360Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3863, "text": "Step 3: Glutaraldehyde-free silver stained gels", "checked": false, "checklist_id": 940, "created_at": "2018-12-20T14:03:52.362Z", "updated_at": "2018-12-20T14:04:56.749Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3864, "text": "Step 4: Coomassie-stained gels", "checked": false, "checklist_id": 940, "created_at": "2018-12-20T14:03:52.369Z", "updated_at": "2018-12-20T14:04:56.761Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 } ] }, "position": 1 } ] }, { "step": { "id": 4825, "name": "Silver stained gels", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-26T13:14:10.347Z", "updated_at": "2018-12-24T06:30:40.360Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nSilver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity, whilst using very simple and cheap equipment and chemicals." }, "position": 0 }, { "checklist": { "checklist": { "id": 916, "name": "Washing:", "step_id": 4825, "created_at": "2018-12-07T10:50:00.146Z", "updated_at": "2018-12-07T11:37:14.463Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3776, "text": "Remove destain solution", "checked": false, "checklist_id": 916, "created_at": "2018-12-07T10:50:00.148Z", "updated_at": "2018-12-07T10:50:00.148Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3777, "text": "Wash gel piece with 400 μL water", "checked": false, "checklist_id": 916, "created_at": "2018-12-07T10:50:00.157Z", "updated_at": "2018-12-20T12:00:27.604Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3778, "text": "Shake for 15 min at room temperature", "checked": false, "checklist_id": 916, "created_at": "2018-12-07T10:50:00.166Z", "updated_at": "2018-12-07T10:50:00.166Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3779, "text": "Repeat wash at least twice or until gel pieces are colourless.", "checked": false, "checklist_id": 916, "created_at": "2018-12-07T10:50:00.175Z", "updated_at": "2018-12-07T10:50:00.175Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 }, { "checklist": { "checklist": { "id": 918, "name": "Dehydration:", "step_id": 4825, "created_at": "2018-12-07T11:36:41.173Z", "updated_at": "2018-12-07T11:37:28.556Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3783, "text": "Dehydrate gel pieces with 400 μL of 100% acetonitrile for 10 min", "checked": false, "checklist_id": 918, "created_at": "2018-12-07T11:36:41.177Z", "updated_at": "2018-12-20T12:00:27.594Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3784, "text": "Remove supernatant", "checked": false, "checklist_id": 918, "created_at": "2018-12-07T11:36:41.204Z", "updated_at": "2018-12-07T11:36:41.204Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3785, "text": "Dry gel pieces in a vacuum centrifuge", "checked": false, "checklist_id": 918, "created_at": "2018-12-07T11:36:41.211Z", "updated_at": "2018-12-07T11:36:41.211Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 2 } ] }, { "step": { "id": 4823, "name": "Excise gel bands", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-26T13:01:32.320Z", "updated_at": "2019-02-20T07:32:22.475Z", "last_modified_by_id": 202, "protocol_id": 3678 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3740, "created_at": "2019-02-20T07:32:07.412Z", "updated_at": "2019-02-20T07:32:22.467Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 12461, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Bottom_up_proteomics.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n

Once you have run your gel, move it to an open UV box (be sure to wear proper UV protection - especially for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Try to get as little excess gel around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important during the DNA purification step, as many kits cannot handle more than a certain total volume of gel per reaction.

" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2262, "name": "Protein preparation: Liquid Chromatography Fraction", "due_date": null, "description": null, "x": 0, "y": 16, "my_module_group_id": 1179, "created_at": "2018-11-26T13:45:56.851Z", "updated_at": "2018-12-19T09:02:54.890Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 1, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5980, "input_id": 2345, "output_id": 2262 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3682, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2262, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-26T13:45:56.859Z", "updated_at": "2019-02-20T07:31:56.209Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4840, "name": "Neutralization of samples", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-26T13:50:47.141Z", "updated_at": "2019-02-20T07:32:22.048Z", "last_modified_by_id": 202, "protocol_id": 3682 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3739, "created_at": "2019-02-20T07:31:55.926Z", "updated_at": "2019-02-20T07:32:22.040Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 12461, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Bottom_up_proteomics.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n
The pH of the sample may be neutralized either prior to or after drying via vacuum centrifugation to remove acetonitrile and trifluoroacetic acid. RP-HPLC samples will likely contain aqueous trifluoroacetic acid and acetonitrile since these are the most commonly used mobile phases. The NH4HCO3 solution should be prepared fresh, as the pH of the solution will increase with age at room temperature. It is possible to store for several days at 4°C, but the pH should be checked prior to use.\n
" }, "position": 0 }, { "checklist": { "checklist": { "id": 944, "name": "To neutralize prior to drying:", "step_id": 4840, "created_at": "2018-12-20T14:20:33.276Z", "updated_at": "2018-12-20T14:20:33.276Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3878, "text": "Add sufficient 1 M NH4HCO3, pH8.5.", "checked": false, "checklist_id": 944, "created_at": "2018-12-20T14:20:33.278Z", "updated_at": "2018-12-20T14:20:33.278Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3879, "text": "Adjust final pH to ~8.0.", "checked": false, "checklist_id": 944, "created_at": "2018-12-20T14:20:33.285Z", "updated_at": "2018-12-20T14:20:33.285Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3880, "text": "Then dry via vacuum centrifugation.", "checked": false, "checklist_id": 944, "created_at": "2018-12-20T14:20:33.292Z", "updated_at": "2018-12-20T14:20:33.292Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 }, { "checklist": { "checklist": { "id": 945, "name": "To neutralize after drying:", "step_id": 4840, "created_at": "2018-12-20T14:21:35.471Z", "updated_at": "2018-12-20T14:21:35.471Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3881, "text": "Resuspend dried protein in 100 mM NH4HCO3, pH 8.5.", "checked": false, "checklist_id": 945, "created_at": "2018-12-20T14:21:35.474Z", "updated_at": "2018-12-20T14:21:35.474Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3882, "text": "Spot an aliquot of the neutralized solution on narrow-range pH paper to validate that neutralization (to pH 8.0) has occurred.", "checked": false, "checklist_id": 945, "created_at": "2018-12-20T14:21:35.481Z", "updated_at": "2018-12-20T14:21:35.481Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 } ] }, "position": 2 } ] }, { "step": { "id": 5148, "name": "Reduction of disulfide bonds and alkyle free cysteine residues", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T12:46:55.270Z", "updated_at": "2019-01-31T08:30:26.220Z", "last_modified_by_id": 202, "protocol_id": 3682 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Reduction of disulfide bonds can be achieved with either dithiothreitol (DTT) or tris (2-carboxyethyl)phosphine hydrochloride (TCEP). DTT works optimally at pH 7 to 9, TCEP is typically more efficient, works at a wider pH range (pH 2 to 11), and has no offensive odour. The concentrations and incubation times for DTT, TCEP, and IAA may vary depending on the amount of protein to be treated. Typical concentrations range from 5 to 10 mM for DTT and TCEP, and 10 to 50 mM for iodoacetamide. Incubation times may range from 5 min to 1 hr for the reduction step and 10 min to 1 hr for the alkylation step. Similarly, the reduction can be performed at room temperature, 37°C or 56°C. Each parameter can be optimized depending on the nature of the protein sample.

\n
Optional: Following alkylation, adjust the sample to 40 mM DTT.
\n" }, "position": 0 }, { "checklist": { "checklist": { "id": 946, "name": "Guidelines:", "step_id": 5148, "created_at": "2018-12-20T14:24:01.663Z", "updated_at": "2018-12-20T14:24:01.663Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3883, "text": "Add DTT to a final concentration of 10 mM or TCEP to a final concentration of 5 mM.", "checked": false, "checklist_id": 946, "created_at": "2018-12-20T14:24:01.666Z", "updated_at": "2018-12-20T14:24:01.666Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3884, "text": "Incubate sample at room temperature with agitation (e.g. vortexing, or shaking), for 30 min with DTT or 10 min with TCEP.", "checked": false, "checklist_id": 946, "created_at": "2018-12-20T14:24:01.680Z", "updated_at": "2018-12-20T14:24:01.680Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3885, "text": "Add iodoacetamide (IAA) to a concentration of 10 mM final and place at room temperature in the dark for 30 min.", "checked": false, "checklist_id": 946, "created_at": "2018-12-20T14:24:01.690Z", "updated_at": "2018-12-20T14:24:01.690Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 } ] }, { "step": { "id": 5149, "name": "Digestion of protein samples", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T12:52:36.364Z", "updated_at": "2019-01-31T08:32:20.691Z", "last_modified_by_id": 202, "protocol_id": 3682 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n
Digest the protein sample into peptides using an appropriate enzyme (e.g., chymotrypsin, trypsin, LysC, or AspN) or chemical (CNBr/70% formic acid). If using trypsin, add sufficient enzyme for final trypsin: protein ratio of 1:20 to 1:100 (w/w). Vortex briefly, seal the tube with Parafilm and incubate with end-over-end rotation at 37°C for 4 to 18 hours.
\n
\n
The amount of trypsin needed will vary depending on the amount of protein sample present and the desired speed of digestion. To minimize trypsin autolysis, which will add extra peaks to the MS spectra, use the minimum amount of trypsin. Typically, a ratio of 1:20 (w/w) is sufficient for complete digestion of 10 μg protein within 4 hr. If protein concentration is especially high or the accessibility of trypsin to the cleavage sites is hampered (e.g., due to insolubility), it may be helpful to add another aliquot of trypsin or perform short digestion (4 hours) with LysC prior to the addition of trypsin. Finally, the activity of trypsin is enhanced in the presence of acetonitrile (10% to 50% v/v), and, thus, acetonitrile may be added to aid in the solubilization of the protein during tryptic digestion.
\n
\n
Trypsin activity is greatest at pH 8.0. Thus, it is recommended that the pH of the solution be checked prior to the addition of the enzyme. In cases where sample volume is small, a 1- to 2-μL sample may be tested using pH paper. Stop reaction by adding 5 μL of 1.0% trifluoroacetic (TFA) acid (pH after TFA addition should be ~2 to 3).
\n" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2346, "name": "TiO2 Enrichment (optional*)", "due_date": null, "description": null, "x": 73, "y": 15, "my_module_group_id": 1179, "created_at": "2018-12-07T14:01:45.181Z", "updated_at": "2018-12-19T09:02:54.895Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 4, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5983, "input_id": 2275, "output_id": 2346 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3837, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2346, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-12-07T14:01:45.189Z", "updated_at": "2019-01-31T08:39:24.163Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5154, "name": "Elution and collection of samples", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T14:20:38.293Z", "updated_at": "2018-12-24T08:06:13.141Z", "last_modified_by_id": 202, "protocol_id": 3837 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Follow elution and collection guidelines below.

" }, "position": 0 }, { "checklist": { "checklist": { "id": 951, "name": "Guidelines:", "step_id": 5154, "created_at": "2018-12-20T14:51:18.465Z", "updated_at": "2018-12-20T14:51:18.465Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3901, "text": "If samples have been dried, then resuspended in 3 μL 1 of % SDS. Dilute IMAC–1% TFA and IMAC-FT samples each in 5 volumes of TiO2 loading solution.", "checked": false, "checklist_id": 951, "created_at": "2018-12-20T14:51:18.467Z", "updated_at": "2018-12-20T14:51:18.467Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3902, "text": "Load each of the samples onto one TiO2 column (for simple samples) or three to four TiO2 columns (for complex mixtures containing ≥120 μg protein).", "checked": false, "checklist_id": 951, "created_at": "2018-12-20T14:51:18.476Z", "updated_at": "2018-12-20T14:51:18.476Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3903, "text": "Collect TiO2 flow through in microcentrifuge tubes. Do this step slowly using Combi-Syringe to apply the pressure.", "checked": false, "checklist_id": 951, "created_at": "2018-12-20T14:51:18.483Z", "updated_at": "2018-12-20T14:51:18.483Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3904, "text": "Wash the TiO2 columns using 5 μL TiO2 loading solution and pool the eluates with the TIO2FT fraction from the previous step. Do this step slowly using Combi-Syringe to apply the pressure.", "checked": false, "checklist_id": 951, "created_at": "2018-12-20T14:51:18.491Z", "updated_at": "2018-12-20T14:51:18.491Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3905, "text": "Elute the phosphopeptides using 30 μL high-pH elution solution. Use 30 μL per column and pool the eluates from the same sample. This step should be performed slowly using a Combi-Syringe to apply pressure.\r\nAcidify the eluates using 100% TFA to a pH of ~2 to 3.", "checked": false, "checklist_id": 951, "created_at": "2018-12-20T14:51:18.499Z", "updated_at": "2018-12-20T14:51:18.499Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] }, "position": 1 } ] }, { "step": { "id": 5156, "name": "Preparation of samples for MS", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T14:34:54.225Z", "updated_at": "2018-12-24T08:07:19.825Z", "last_modified_by_id": 202, "protocol_id": 3837 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Different preparations of samples for different types of mass spectrometry. 

" }, "position": 0 }, { "checklist": { "checklist": { "id": 920, "name": "For MALDI-TOF-MS/MS:", "step_id": 5156, "created_at": "2018-12-07T14:34:54.228Z", "updated_at": "2018-12-07T14:34:54.228Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3789, "text": "Elute the peptides from Oligo R3 column using 5 μL phosphopeptide RP elution buffer", "checked": false, "checklist_id": 920, "created_at": "2018-12-07T14:34:54.230Z", "updated_at": "2018-12-20T12:16:38.311Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3790, "text": "Spot directly onto MALDI sample plate, using a pipet.", "checked": false, "checklist_id": 920, "created_at": "2018-12-07T14:34:54.239Z", "updated_at": "2018-12-07T14:34:54.239Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3791, "text": "As spot is drying, if matrix crystals are not forming well, spot an additional 0.5 μL phosphopeptide RP elution buffer.", "checked": false, "checklist_id": 920, "created_at": "2018-12-07T14:34:54.248Z", "updated_at": "2018-12-20T12:16:38.318Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 }, { "checklist": { "checklist": { "id": 921, "name": "For LC-ESI-MS/MS:", "step_id": 5156, "created_at": "2018-12-07T14:34:54.261Z", "updated_at": "2018-12-07T14:34:54.261Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3792, "text": "Elute the peptides from the Oligo R3 column using 30 μl phosphopeptide RP elution buffer.", "checked": false, "checklist_id": 921, "created_at": "2018-12-07T14:34:54.263Z", "updated_at": "2018-12-07T14:34:54.263Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3793, "text": "Dry pooled eluates by vacuum centrifugation.", "checked": false, "checklist_id": 921, "created_at": "2018-12-07T14:34:54.271Z", "updated_at": "2018-12-07T14:34:54.271Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3794, "text": "Resuspend the monophosphopeptides in 0.5 μL 100% formic acid and add 10 μl of 0.1% formic acid immediately.", "checked": false, "checklist_id": 921, "created_at": "2018-12-07T14:34:54.278Z", "updated_at": "2018-12-20T12:16:38.303Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 2 } ] }, { "step": { "id": 5155, "name": "Preparation of a slurry of Oligo R3 reversed-phase resin", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T14:31:41.630Z", "updated_at": "2019-01-31T08:39:07.689Z", "last_modified_by_id": 202, "protocol_id": 3837 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n
Prepare a slurry of Oligo R3 reversed-phase resin in 50% acetonitrile. Pack the Oligo R3 into a P20 narrow-bore pipet tip. Squeeze the tip of the narrow-bore P20 pipet tip to prevent the beads from leaking out. Sufficient Oligo R3 slurry should be added to make the columns ~5 mm long. It is important to have a good seal on these columns, as column breakthrough will block on-line liquid chromatography systems.
\n
Load each of the phosphopeptide-containing eluates that have been enriched with the TiO2 strategy onto one (for simple samples) or two to three (for complex mixtures) Oligo R3 columns depending on the amount of material.
Wash the Oligo R3 columns using 30 μL 0.1% TFA.
\n" }, "position": 0 } ] }, { "step": { "id": 5153, "name": "Preparation of TiO2 bead slurry", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T14:15:28.126Z", "updated_at": "2019-01-29T10:23:10.142Z", "last_modified_by_id": 202, "protocol_id": 3837 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n
Prepare a TiO2 bead slurry in 100% acetonitrile. Pack the TiO2 in StageTips that have been pre-packed with C8 disks. The resulting TiO2 columns should be ~4 to 5 mm long.
\n
TiO2 is light sensitive, so keep the powder in an amber or dark glass container or in a microcentrifuge tube covered with aluminium foil. When not in use, TiO2 should be in a container stored in a dark place (e.g., a drawer). It should not be exposed to light for longer than necessary.
\n" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2345, "name": "IMAC Phosphopeptide Enrichment (optional*)", "due_date": null, "description": null, "x": 38, "y": 15, "my_module_group_id": 1179, "created_at": "2018-12-07T13:34:00.077Z", "updated_at": "2018-12-19T09:02:54.880Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 3, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5979, "input_id": 2346, "output_id": 2345 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3836, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2345, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-12-07T13:34:00.086Z", "updated_at": "2019-01-25T12:07:01.449Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5152, "name": "Elution and collection", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T13:58:12.351Z", "updated_at": "2018-12-20T14:48:45.965Z", "last_modified_by_id": 202, "protocol_id": 3836 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
Dry each eluate containing the multiply phosphorylated peptides, monophosphopeptides, and the IMAC flow through separately using vacuum centrifugation.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 950, "name": "Guidelines:", "step_id": 5152, "created_at": "2018-12-20T14:48:32.247Z", "updated_at": "2018-12-20T14:48:32.247Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3897, "text": "Collect the column flow through in microcentrifuge tubes.", "checked": false, "checklist_id": 950, "created_at": "2018-12-20T14:48:32.249Z", "updated_at": "2018-12-20T14:48:32.249Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3898, "text": "Wash the IMAC column with 50 μL IMAC wash solution and pool the eluate with the fraction from the previous step. This need to be done slowly using a Combi-Syringe to apply pressure.", "checked": false, "checklist_id": 950, "created_at": "2018-12-20T14:48:32.256Z", "updated_at": "2018-12-20T14:48:32.256Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3899, "text": "Elute and collect the monophosphopeptides using 50 μl low-pH elution solution. This needs to be done quickly using a Combi-Syringe to apply pressure.", "checked": false, "checklist_id": 950, "created_at": "2018-12-20T14:48:32.263Z", "updated_at": "2018-12-20T14:48:32.263Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3900, "text": "Elute and collect the multiply phosphorylated peptides using 70 μL high-pH elution solution. This step should be performed slowly using a Combi-Syringe to apply pressure.", "checked": false, "checklist_id": 950, "created_at": "2018-12-20T14:48:32.269Z", "updated_at": "2018-12-20T14:48:32.269Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 } ] }, { "step": { "id": 5151, "name": "Add protein sample to IMAC beads", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T13:50:12.807Z", "updated_at": "2019-01-25T12:07:01.391Z", "last_modified_by_id": 202, "protocol_id": 3836 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Squeeze the tip of the narrow-bore pipet tip nearly shut, as this will prevent bead loss. The Combi-Syringe is only to be used for the generation of pressure in the column, not for measuring and loading the aliquots. The Combi-Syringe is reusable and should therefore not come into contact with any solution.

\n
 
\n" }, "position": 0 }, { "checklist": { "checklist": { "id": 949, "name": "Guideline:", "step_id": 5151, "created_at": "2018-12-20T14:47:07.628Z", "updated_at": "2018-12-20T14:47:07.628Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3894, "text": "Add protein sample to the IMAC beads in a volume of IMAC wash solution that is 10 times the volume of the packed beads.", "checked": false, "checklist_id": 949, "created_at": "2018-12-20T14:47:07.630Z", "updated_at": "2018-12-20T14:47:07.630Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3895, "text": "Incubate with constant, gentle agitation, at room temperature for 30 to 60 min.", "checked": false, "checklist_id": 949, "created_at": "2018-12-20T14:47:07.638Z", "updated_at": "2018-12-20T14:47:07.638Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3896, "text": "Using a Combi-Syringe and gentle pressure, pack the IMAC beads in a narrow bore tip by gradually adding the bead slurry in 20 μL aliquots.", "checked": false, "checklist_id": 949, "created_at": "2018-12-20T14:47:07.654Z", "updated_at": "2018-12-20T14:47:07.654Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 } ] }, { "step": { "id": 5150, "name": "Wash IMAC beads", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-07T13:34:50.552Z", "updated_at": "2018-12-20T14:44:55.371Z", "last_modified_by_id": 202, "protocol_id": 3836 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Too many IMAC beads may result in nonspecific binding of peptides because of reduced washing efficiency. A small amount of wash solution should be left in the tube with the beads, to avoid aspirating the beads.\n

\n
 
\n" }, "position": 0 }, { "checklist": { "checklist": { "id": 948, "name": "Guideline:", "step_id": 5150, "created_at": "2018-12-20T14:44:55.374Z", "updated_at": "2018-12-20T14:44:55.374Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3891, "text": "Wash IMAC (immobilized metal affinity chromatography) beads. You will need 5 to 7 μL for simple mixtures and 30 to 50 μL for complex mixtures containing ≥120 μg protein.", "checked": false, "checklist_id": 948, "created_at": "2018-12-20T14:44:55.376Z", "updated_at": "2018-12-20T14:44:55.376Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3892, "text": "Applying 50 μL IMAC wash solution, vortex and centrifuge at low speed (e.g., 800 rpm in a benchtop microcentrifuge) at room temperature to pellet the beads.", "checked": false, "checklist_id": 948, "created_at": "2018-12-20T14:44:55.383Z", "updated_at": "2018-12-20T14:44:55.383Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3893, "text": "Remove the supernatant by pipetting.", "checked": false, "checklist_id": 948, "created_at": "2018-12-20T14:44:55.390Z", "updated_at": "2018-12-20T14:44:55.390Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2274, "name": "Protein preparation: Affinity Capture", "due_date": null, "description": null, "x": 0, "y": 32, "my_module_group_id": 1179, "created_at": "2018-11-27T17:13:44.860Z", "updated_at": "2018-12-19T09:02:54.892Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 0, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5981, "input_id": 2345, "output_id": 2274 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3704, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2274, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-27T17:13:44.867Z", "updated_at": "2019-02-20T07:31:43.409Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4872, "name": "Elute protein from the matrix", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:40:37.750Z", "updated_at": "2018-12-24T07:26:44.813Z", "last_modified_by_id": 202, "protocol_id": 3704 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
Down below are the guidelines for elution of proteins.  

\n
" }, "position": 0 }, { "checklist": { "checklist": { "id": 858, "name": "Guidelines:", "step_id": 4872, "created_at": "2018-11-27T17:40:37.753Z", "updated_at": "2018-12-20T14:36:53.748Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3564, "text": "Use a 5× volume of 0.1 M glycine, pH 2.5.", "checked": false, "checklist_id": 858, "created_at": "2018-11-27T17:40:37.755Z", "updated_at": "2018-11-27T17:40:37.755Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3565, "text": "Apply the elution buffer and allow the buffer to thoroughly wet the matrix for 5 to 10 min.", "checked": false, "checklist_id": 858, "created_at": "2018-11-27T17:40:37.764Z", "updated_at": "2018-11-27T17:40:37.764Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3566, "text": "Collect the eluate.", "checked": false, "checklist_id": 858, "created_at": "2018-11-27T17:40:37.772Z", "updated_at": "2018-11-27T17:40:37.772Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3567, "text": "To enhance recovery, wash the matrix with an additional 5× volume of 0.1 M glycine, pH 2.5.", "checked": false, "checklist_id": 858, "created_at": "2018-11-27T17:40:37.780Z", "updated_at": "2018-11-27T17:40:37.780Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3568, "text": "Adjust eluate to neutral pH by addition of an equal volume of 200 mM NH4HCO3.", "checked": false, "checklist_id": 858, "created_at": "2018-11-27T17:40:37.788Z", "updated_at": "2018-11-27T17:40:37.788Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] }, "position": 1 } ] }, { "step": { "id": 4873, "name": "Reduction of disulfide bonds", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:42:41.887Z", "updated_at": "2018-12-24T07:32:38.099Z", "last_modified_by_id": 202, "protocol_id": 3704 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nDisulfide (sulfur-sulfur) linkages between two cysteine residues are an integral component of the three-dimensional structure of many proteins. Disulfide reducing agents are routinely used in biochemical reactions for peptides and proteins prior to MS analysis. " }, "position": 0 }, { "checklist": { "checklist": { "id": 859, "name": "To do:", "step_id": 4873, "created_at": "2018-11-27T17:42:41.891Z", "updated_at": "2018-11-27T17:42:41.891Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3569, "text": "Add DTT to a final concentration of 10 mM or TCEP to a final concentration of 5 mM.", "checked": false, "checklist_id": 859, "created_at": "2018-11-27T17:42:41.894Z", "updated_at": "2018-11-27T17:42:41.894Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3570, "text": "Incubate sample at room temperature with vortexing for 10 min with TCEP or 30 min with DTT.", "checked": false, "checklist_id": 859, "created_at": "2018-11-27T17:42:41.915Z", "updated_at": "2018-11-27T17:42:41.915Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3571, "text": "Next, to alkylate free cysteines, add iodoacetamide to a final concentration of 10 mM", "checked": false, "checklist_id": 859, "created_at": "2018-11-27T17:42:41.923Z", "updated_at": "2018-11-27T17:42:41.923Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3572, "text": "Place at room temperature in the dark for 30 min.", "checked": false, "checklist_id": 859, "created_at": "2018-11-27T17:42:41.934Z", "updated_at": "2018-11-27T17:42:41.934Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 } ] }, { "step": { "id": 4874, "name": "Digestion of the protein sample", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:43:58.146Z", "updated_at": "2018-12-24T07:33:39.499Z", "last_modified_by_id": 202, "protocol_id": 3704 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 947, "name": "Guidelines:", "step_id": 4874, "created_at": "2018-12-20T14:41:18.229Z", "updated_at": "2018-12-20T14:41:18.229Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3886, "text": "Use an appropriate enzyme or chemical. If using trypsin, add sufficient enzyme for final trypsin: protein ratio of 1:20 to 1:100 (w/w).", "checked": false, "checklist_id": 947, "created_at": "2018-12-20T14:41:18.231Z", "updated_at": "2018-12-20T14:41:18.231Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3887, "text": "Vortex briefly.", "checked": false, "checklist_id": 947, "created_at": "2018-12-20T14:41:18.238Z", "updated_at": "2018-12-20T14:41:18.238Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3888, "text": "Seal the tube with Paraffin.", "checked": false, "checklist_id": 947, "created_at": "2018-12-20T14:41:18.244Z", "updated_at": "2018-12-20T14:41:18.244Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3889, "text": "Incubate with end-over-end rotation at 37°C for 4 to 18 hours.", "checked": false, "checklist_id": 947, "created_at": "2018-12-20T14:41:18.250Z", "updated_at": "2018-12-20T14:41:18.250Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3890, "text": "Stop reaction by adding 5 μL of 1.0% trifluoroacetic (TFA) acid.", "checked": false, "checklist_id": 947, "created_at": "2018-12-20T14:41:18.257Z", "updated_at": "2018-12-20T14:41:18.257Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] }, "position": 1 } ] }, { "step": { "id": 4871, "name": "Wash affinity matrix", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:15:38.260Z", "updated_at": "2019-02-20T07:32:21.681Z", "last_modified_by_id": 202, "protocol_id": 3704 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3738, "created_at": "2019-02-20T07:31:43.019Z", "updated_at": "2019-02-20T07:32:21.672Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 12461, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Bottom_up_proteomics.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n
\nWash affinity matrix containing the adsorbed protein sample with the wash buffer such as PBS to elute the unbound or nonspecifically bound proteins from the matrix. Beads may be washed by resuspending them in the wash buffer with gentle vortexing, then microcentrifuging at low speed (e.g., 800 rpm in a standard microcentrifuge) at room temperature to pellet the beads, and removing the supernatant by pipetting.
If detergent is present in the starting material, it may be necessary to increase the number of washes to remove residual detergent. Often this can be difficult and may require up to ten additional 20× volume washes (i.e., 20 times the packed volume of the matrix). For most immunocapture methods, PBS is an effective wash buffer, but other neutral-pH saline buffers such as HEPES or Tris·Cl can be used. If greater stringency is required, for example, a high-salt wash or inclusion of a chaotropic agent, perform more stringent washes first followed by additional PBS washes. It is often necessary to optimize wash conditions for individual affinity-capture methods.
" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2275, "name": "Peptide Sample Cleanup", "due_date": null, "description": null, "x": 73, "y": 32, "my_module_group_id": 1179, "created_at": "2018-11-27T17:52:31.026Z", "updated_at": "2018-12-19T09:02:54.882Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 5, "experiment_id": 454, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5982, "input_id": 2276, "output_id": 2275 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3705, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2275, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-27T17:52:31.033Z", "updated_at": "2019-01-31T08:40:47.434Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4876, "name": "Prepare the column", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:54:41.488Z", "updated_at": "2018-11-27T17:57:19.111Z", "last_modified_by_id": 202, "protocol_id": 3705 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
 The processing of samples can be performed using a pipet or centrifuge, or with the aid of a vacuum manifold.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 860, "name": "To do:", "step_id": 4876, "created_at": "2018-11-27T17:57:03.419Z", "updated_at": "2018-11-27T17:57:03.419Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3573, "text": "Wash the column three times with 200 μl 100% acetonitrile.", "checked": false, "checklist_id": 860, "created_at": "2018-11-27T17:57:03.421Z", "updated_at": "2018-11-27T17:57:03.421Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3574, "text": "Discard flow through.", "checked": false, "checklist_id": 860, "created_at": "2018-11-27T17:57:03.430Z", "updated_at": "2018-11-27T17:57:03.430Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3575, "text": "Rinse the column three times with 200 μL 0.1% TFA.", "checked": false, "checklist_id": 860, "created_at": "2018-11-27T17:57:03.437Z", "updated_at": "2018-12-20T12:17:19.310Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3576, "text": "Discard flow through.", "checked": false, "checklist_id": 860, "created_at": "2018-11-27T17:57:03.445Z", "updated_at": "2018-11-27T17:57:03.445Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 } ] }, "position": 1 } ] }, { "step": { "id": 4879, "name": "Elute peptides from the column", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T18:01:53.039Z", "updated_at": "2019-01-31T08:40:47.389Z", "last_modified_by_id": 202, "protocol_id": 3705 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Use 30 μL of RP LC-ESI-MS/MS elution solution for samples that will be analyzed. Collect the eluate in a clean tube. The samples are now ready for MS analysis.

" }, "position": 0 } ] }, { "step": { "id": 4878, "name": "Rinse column", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T18:00:46.914Z", "updated_at": "2019-01-29T10:24:17.412Z", "last_modified_by_id": 202, "protocol_id": 3705 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
\nThree to ten times with 200 μL 0.1% TFA. The volume required for sufficient rinsing depends on the concentration of salts and other reagents in the sample. If electrospray/nanospray is to be performed, it is advisable to subsequently rinse the column three times with 200 μL water to remove the TFA, which may interfere with ionization in electrospray/nanospray.
" }, "position": 0 } ] }, { "step": { "id": 4877, "name": "Application of samples", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-27T17:59:44.389Z", "updated_at": "2018-12-24T08:07:53.994Z", "last_modified_by_id": 202, "protocol_id": 3705 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Follow guidelines for sample preparation. 

" }, "position": 0 }, { "checklist": { "checklist": { "id": 861, "name": "Preparation of samples:", "step_id": 4877, "created_at": "2018-11-27T17:59:44.392Z", "updated_at": "2018-11-27T17:59:44.392Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3577, "text": "Add 10% TFA to bring the final concentration to 0.1% TFA to the peptide sample.", "checked": false, "checklist_id": 861, "created_at": "2018-11-27T17:59:44.394Z", "updated_at": "2018-11-27T17:59:44.394Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3578, "text": "Apply the sample to the column", "checked": false, "checklist_id": 861, "created_at": "2018-11-27T17:59:44.403Z", "updated_at": "2018-11-27T17:59:44.403Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3579, "text": "Use a pipette to push sample through slowly", "checked": false, "checklist_id": 861, "created_at": "2018-11-27T17:59:44.410Z", "updated_at": "2018-11-27T17:59:44.410Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 } ] } ] } ], "results": [ ] } ], "my_module_groups": [ { "id": 1179, "created_at": "2018-12-19T09:02:54.877Z", "updated_at": "2018-12-19T09:02:54.877Z", "created_by_id": 202, "experiment_id": 454 } ] }