Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.


Current content of STAMPS
Information about present pathways and the pathway structure including the protein and metabolite node location and connection were initially extracted from KEGG database 1 , UniProt 2 and NCBI 3 .   The time evaluation is based on a domain expert using all mentioned tools and frameworks to create a fasta file and a spectral library containing all necessary protein information of the insulin signaling pathway. Note, that for PeptideAtlas & NIST library only complete spectral libraries for mouse could be downloaded as well as a complete library was created when using raw files from ProteomicsDB. In Picky, only ≤ 100 IDs can be used in one assay. When only human experiments were available in those databases, we took human instead of mouse data. human curation of spectra X n/a n/a n/a n/a pathway curation by domain experts X statistical support for method dev. X X web-editor for editing pathways X high-resolution spectra X X downloadable spectra

Feature comparison
proteins tissue resolved X X proteins chromosome resolved X X X proteins pathway resolved X visual protein selection X protein batch selection X X proteins enzymatically resolved X X proteins subcellular resolved X X assay development X X integration with cons. analysis tool * X X (X) = available in the next version n/a = information not available *e.g. Skyline

Sample preparation
Cell pellets were lysed with a 1% SDS lysis buffer. Frozen mouse organs were homogenized by grinding in liquid nitrogen, one at a time. To achieve this, each organ were placed in a liquid nitrogen-cooled ceramic mortar separately. The tissues were manually grinded with a pestle to obtain a fine powder (sterile conditions). The powder was collected in Eppendorf reaction tubes and 50 µg of each prepared organ were lysed using a 1% SDS lysis buffer.
To remove interfering DNA, the samples were treated with Benzonase® for 30 min at 37 °C, followed by protein precipitation using 3 volumes of ice cold acetone overnight at -20 °C.

Method development and targeted LC-MS analysis
To develop the method for the targeted LC-MS/MS analysis, we used STAMPS to select proteins and peptides for the tricarboxylic acid (TCA) cycle and insulin signaling pathway according to the pathway browser and the built spectral library. After importing the method into Skyline, three transitions were chosen for each peptide that showed the best signal to noise ratio. In addition, synthetic peptides were synthesized for each chosen proteotypic peptide to avoid false positive peak annotation and to confirm the correct retention time.

Application -Results
To determine the practicability of the workflow, we compared STAMPS with state-of-the-art targeted proteomics tools (Table S4). We measured the time to create a targeted assay for insulin-signaling. Here, a time reduction of ⪆ 150-fold was achieved compared to the second fastest tool. A list of all features in comparison to other state-of-the-art tools is shown in Table   S5.
Next, to confirm the applicability of STAMPS, we selected insulin-signaling and the tricarboxylic acid cycle (TCA), two pathways important for the overall metabolism of higher S9 eukaryotes. Key proteins of these pathways were analyzed after inducing insulin resistance and sensitivity (Figure 2, S1 -S3).

Tutorials
Creating an assay using the pathway browser One of the major tasks in targeted proteomics is building up methods to analyze proteins of one or multiple pathways to uncover their behavior upon different treatments revealing coherencies and patterns, for instance in the metabolism. To facilitate those studies and gain results as shown in Figure 2, STAMPS provides a multitude of intuitive interfaces for fast protein selection, spectra reviewing, assay building and is interlinked with Skyline.

1) Select pathway browser
The first page of the STAMPS 5 website is leading to all main protein selection options as visible in Figure S4. Clicking on "Pathway Browser" in the lower panel will lead to the interactive main pathway browser window ( Figure S5). The view can be shifted (right click) and the zoom can be changed (mouse wheel or magnifier icons on bottom right position).
The metabolic pathways are plotted in a graph-based fashion. Metabolites are presented as circles; proteins which act in the same metabolic reaction are grouped in protein boxes.
By directly clicking on metabolite or protein labels, additional information is displayed.
Links to other Pathways are indicated in round-cornered boxes. By clicking on them, the according pathway will be opened.

3) Select proteins
Proteins of choice can be (de)selected by clicking on their checkboxes. Double-clicking on a protein group box will (de)select all proteins of the according box. By clicking and holding down the left mouse button on a free space and moving the mouse, a protein selection area will be spanned. When releasing the button, all proteins within this selection window will be (de)selected.

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The user has the possibility to get a statistical overview for each pathway by clicking on the arrows in the upper right corner. A window will appear giving information of the total protein number of the selected pathway, protein coverage, number of selected proteins and additional information regarding the number of peptides, spectra and validation (see Figure S6).

5) Check the protein selection and review spectra
Having selected all proteins for an assay, the user can review the selection by clicking on "Check spectra" in the upper panel. A new window opens as shown in Figure S7. On the left-hand side, a table is showing all selected proteins. The user has the possibility to expand each protein to show its corresponding peptides that are stored with an according spectrum in the database. By clicking on the precursor mass, the corresponding spectrum pops up showing y and b ions. All proteins, peptides or spectra can be (de)selected on this screen for the final assay by checking or unchecking the blue boxes. To refine the assay even more, specific filter criteria can be applied. For instance, the user can filter for peptide length and precursor charge as well as choose between variable and fixed modifications of the oxidation of methionine and/or the carbamidomethylation of cysteine. In Addition, there is the possibility to filter for specific tissues. The user has to take into consideration that only proteins, peptides and precursors will be displayed, S14 which were identified and stored in our database and in addition satisfy the adjusted filter criteria.

6) Download the assay
Once the selection is done, the user can proceed to download the assay by clicking on "Go to download" on the bottom right side of the dialog. A spectral library in .blib format, a fasta file containing all selected protein sequences and a Skyline project file are being created on the fly and packed as a .zip file offered for download. After downloading and unzipping, the Skyline project file can easily be opened in Skyline 6 with the complete assay ready to start the targeted analyses.

Figure S7 | Review dialog for (de)selecting proteins, peptides or single spectra in the assay.
Creating an assay using the accession list

1) Open the protein selection window via accession IDs
For this step, a list of UniProt 7 accession IDs is required. On the main page (consider Figure S4) one has to click on "Accession Search" in the lower panel.

2) Paste your protein accession IDs
A window for inserting the accession IDs is being opened (as seen in Figure S8). The drop down menu is used to select the desired species. The user can type in the accession IDs into the text field. If there is a longer list, it is possible to copy and paste it into the text field. Click on "Next" to proceed.

3) Check the protein selection and review the spectra
The review dialog appears as known from the previous instructions ( Figure S7), proceeding with step 5 of the pathway browser tutorial.

Figure S8 | Dialog for selecting proteins via a list of UniProt accession IDs.
Creating an assay using the chromosome browser

1) Open protein selection window via chromosome browser
Another way of building up assays for targeted LC-MS/MS analysis is by selecting proteins of interest according to their gene location on the chromosomes. On the main page (consider Figure S4) click on "Chromosome Search" in the lower panel.

2) Paste your protein accession IDs
A window for selecting genes within the chromosomes appears (as illustrated in Figure   S9) On the right-hand side, a gene table lists all registered genes available in the particular chromosome region including gene name and accession number. Genes can be (de)selected with the checkboxes to the right. When the selection is complete, one can proceed by clicking on "Next". Only proteins and peptides according to the selected genes that were identified and stored in the background library will be displayed.

3) Check your protein selection and review spectra
The review dialog appears as known from the previous instructions ( Figure S7), proceeding with step 5 of the pathway browser tutorial.

Figure S9 | Chromosome browser for selecting proteins within regions of chromosomes.
Using the pathway editor to create / update / delete pathways

1) Open the pathway editor
Open the editor 8 and type in the credentials. The editor is split into a top menu strip, a tool box with all functions such as buttons on the left hand side and the current view (canvas) on the pathway on the right hand side (consider Figure S10).

Figure S10 | Basic view on the pathway editor.
The toolbox itself is split into two types of functions i) for creating visual entities (protein nodes, metabolite nodes, edges, etc.) and ii) for alternating the entities (highlight labels, move entities, etc.). The pathway to be edited can be selected via the buttons "Metabolite Pathways" or "Signaling Pathways" as well as species, respectively.

2) Creating a new pathway
To create a new pathway which is not yet registered in the database, click on "Manage entries". In the opening window (consider Figure S11), the user can choose to work on pathway group, single pathway, protein or metabolite entries. Pathway entries contain three attributes, i) the pathway name, ii) the membership to a pathway group and iii) a flag if the pathway is a signaling pathway or not. When clicking at the bottom on "New pathway", a dialog is opening requesting those attributes. For instance, one can type in: pathway name = "UPR Signaling", pathway group = "Other pathway", signaling = checked.
After adding a pathway, its attributes can be furthermore edited in the "Manage entries" window ( Figure S11).

3) Inserting entities into the pathway
Be aware that a recently created pathway is (obviously) empty. Therefore, nothing is visible when selecting the pathway via "Metabolite Pathways" or "Signaling Pathways".
Furthermore, in the regular STAMPS pathway browser ( Figure S5), empty pathways are not selectable. When clicking on "Protein" in the "Create entity" section of the toolbox, the protein node creation mode is active. Moving over the canvas, an empty protein node is being displayed. When clicking on an arbitrary position on the workspace, a protein node is being spotted on this position and the user can continue to create another protein node.
The mode can be deactivated by either clicking once again on "Protein" in the "Create entity" section or selecting another function. Creating pathway nodes, metabolite nodes, labels, or membranes works the same way ( Figure S12).
In the next step, the nodes can be filled with entries from the database. Active modes have to be deactivated before. Now, the user can simply click on the nodes and an according pathway, protein, or metabolite selection window appears. Note, that pathway and metabolite nodes are single nodes whereas multiple proteins can be assigned to one protein node. After assigning entries to the node entities, one can rotate the metabolite

Figure S11 | Window for inserting / editing / deleting entries (as pathways, proteins, metabolites) in the database.
S19 labels by activating the "Rotate metabolite label" mode and clicking on the according metabolite.

Figure S12 | View on the pathway editor canvas after inserting certain functional nodes.
Figure S13 | View on the pathway editor canvas after inserting entries into the according entities.

4) Drawing edges and alter entities
To draw edges, "Edges" mode in the "Create entity" section must be activated first.
Subsequently, anchor points appear for all visual nodes in the canvas. By clicking on one anchor point, holding the mouse button, moving the mouse and releasing over another anchor point, an edge will be drawn and routed automatically. It is allowed to draw edges from each anchor point to each other anchor point, except connecting two anchors of the same node.

Figure S14 | View on the pathway editor canvas after inserting and decorating edges.
By default, the edges are solid and both their endings are not decorated. When clicking on "Change edge type" and clicking on a certain edge the appearance of the edge is changing with each click. In this way one can create solid and dashed edges as well as add arrows and other symbols on the end of each edge. Figure S14 shows an example. Additional functions are moving node entities and highlighting either metabolite or standalone labels.
The last function offers deleting either edges or nodes. When deleting a node, all connected edges will be deleted as well.

Statistics
We added several statistical overviews on different levels comprising spectral, protein, and pathway level. Overviews can be accessed over the main page of STAMPS and are dynamically adjustable. The data is taken directly from the protein and spectral database and the figures are always generated on the fly. Figure S15 illustrates all statistical overviews. Shown are e.g.
normalized distributions of protein abundances over pathways or over tissues, absolute number of proteins, the normalized spectrum abundance factor, protein isoelectric point versus the protein masses as well as the mass residual distribution between the theoretical spectra and the actually measured precursors.

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Figure S15 | Statistical overviews. All figures are interactive allowing the user to change parameters.