Launching the GUI

Launch the graphical interface by double-clicking the SynapSpec application icon:

• Linux: Double-click SynapSpec in Applications folder
• macOS: Double-click SynapSpec in Applications folder
• Windows: Double-click SynapSpec.exe in the installation folder

Interface Overview

Four places, named in the header:

Section	What it is for
Home	Start an analysis, and see what the machine has to run it with
New Analysis	Configure files, search settings, output, and compute options on one page
Runs	What is running, what is queued, what has finished
Templates	Saved settings you can load into a new analysis

A run in progress opens on its own page from Runs. The header carries a count beside Runs when anything is queued.

New Analysis

New Analysis keeps the complete setup on one page. The left rail jumps between Files, Library, Search, Output, and Compute, while the live summary on the right shows what will run and highlights anything that still needs attention. Click an item in the summary to move directly to its setting.

Use the search box in the rail to find a setting. Modified only narrows the page to values that differ from the defaults. The toolbar contains Import, Export, Reset Analysis, and Save as Template.

Files

MS Files. Click Select Input Files and choose your mass spectrometry data (.raw, .mzML, or .wiff). Each file becomes a row where you enter its Condition and Replicate, and where SynapSpec shows the Instrument it detected from the file. Bruker .d data is a directory rather than a file, so it has its own button, Add .d Directories.

Sequence Database. Choose one of the two:

• FASTA Files — a protein sequence database search
• Library File — a spectral library search (.tsv or .txt)

The two are exclusive; picking one replaces the file list beside it.

Sample metadata for differential analysis

Differential analysis uses the metadata entered for each MS file:

• Condition: the experimental group, such as control or treated.
• Replicate: the biological replicate number. Use a positive whole number such as 1, 2, 3.

Technical runs from the same biological replicate should carry the same condition and replicate number. Before running differential analysis, fill metadata for every file, use at least two conditions with at least two biological replicates each, and make sure every input file has a unique filename.

Library

Library settings include enzyme, cleavage mode, search strategy, missed cleavages, modifications, precursor length and charge ranges, and fragment types. This section appears when FASTA files are used; an imported spectral library already defines these properties.

Selecting Custom as the enzyme opens a residue-pair matrix. A selected cell means that the protein is cleaved between those two residues. Row and column headers select an entire group, and the sequence preview updates immediately to show the resulting peptides.

Both ranges are two boxes, a low and a high. Precursor Length Range is the shortest and the longest peptide; Precursor Charge Range is the lowest and the highest charge state.

Setting	Description
Include initiator methionine loss	Also generate peptides after N-terminal initiator methionine removal. On by default.

Search

Search settings contain precursor and protein FDR thresholds, MBR, and the Search Mode that switches between conventional and glycoproteomic analysis.

Output

Output Directory — click Select Folder to choose where results are written.

Queue item name — the name this analysis carries in the Runs list. It is filled in for you from the file count and the date.

Output Format — minimum correlation, file format (.csv, .tsv, .parquet), precursor quantification method, and whether to keep decoys.

Differential Analysis

Setting	Description
Enable Differential Analysis	Performs pairwise comparisons between the conditions defined under Files.
Condition-aware Precursor Correction	Refines precursor MS2 quantities within each condition using protein-level information, giving better quantities for differential analysis.
Quantity Level	The level used for the comparison: Protein Group, Precursor, or Peptide. Protein Group is the default.

When differential analysis is enabled, SynapSpec models biological replicates, combines technical runs belonging to the same replicate, and produces pairwise contrast statistics with report-ready quantity matrices.

Compute

Compute settings control CPU cores, hardware acceleration, and logging. Hardware acceleration is disabled when the machine has no supported device, and the reason is shown beside the switch.

The live summary reports missing required values and warnings, such as an output directory that already contains results. The actions remain available beside the summary:

• Run Now — start immediately.
• Add to Queue — put it in line and set up the next one.

While an analysis is running, Run Now is unavailable because only one analysis runs at a time. Everything else stays open: build the next analysis, clone a finished run, load a template, and add more analyses to the queue.

Runs

One page for every analysis, in three sections.

Currently Running — the analysis in flight. It reports the stage the engine has reached, how long it has been going, and how recently the engine last spoke.

Queued — what will run next, in order. A queued analysis can run next immediately, be edited, move up or down, or be removed. Start Queue runs queued analyses in order; Clear Queue empties the list.

Finished — completed, cancelled, interrupted and failed runs, each with its status.

A finished run

Selecting a run opens its own page. It reports identification and quantification counts for precursors, peptides, and protein groups. Multi-file analyses also show the three identification counts for each raw file. The page keeps the saved counts available when an older output directory has moved or is no longer present.

Use Open Folder to open the output directory and View Details to inspect timestamps and the configuration used for the run. The run log remains available for completed and previous runs.

Clone starts a new analysis from this run’s configuration. The New Analysis page opens with its files, database, and settings already filled in, ready to change whatever you want to vary.

A run still going shows its stage and progress in the same place; the counts appear when the analysis has written its tables.

Templates

A template is a saved set of analysis settings without the files. Load puts one into a new analysis; Export writes it to disk and Import reads one back, which is how a template moves between machines.

Create New opens the template editor. From New Analysis, Save as Template stores the current settings directly. Existing templates can also be edited or deleted from the Templates page.

Glycoproteomic Analysis

SynapSpec identifies N-glycopeptides through the Search Mode setting under Search.

Mode	Description
Conventional	Standard proteomics search (default)
Glyco Proteomics	Enables N-glycopeptide identification

Note: Only N-glycosylation is supported. O-glyco and other glycan types are not available.

Selecting Glyco Proteomics reveals an N-Glyco Configuration panel with two settings.

Allowed Precursor Charges — the charge states considered during glycopeptide identification. It is a row of the numbers 1 to 10 rather than two boxes: click one end of the span you want, then the other, and every number between them lights up. Nothing changes until the second click. Click the same number twice for a span of one. The default is 3 to 7. Glycopeptides carry higher charge states than ordinary peptides because they are larger; widen or narrow the range to match your experiment.

Max Missed Cleavages — the maximum number of missed tryptic cleavage sites allowed for glycopeptide candidates. Accepts 0 or more; the default is 2. This is independent of the missed cleavages setting in Library Generation Settings, which applies to the conventional search only.

Output Results

After the analysis completes, SynapSpec writes its result files to the output directory you chose. See Output Files for what each one contains.