Table of Contents
STEP 1
This tool can be either used in a LIMS or stand-alone. In the stand-alone mode you should either drag/drop your experimental spectrum as a tab-delimited text file or copy paste it (CTRL-V) while moving your mouse over the drop zone. The list of available spectra will be displayed in the table and you can simply click on the one you want to display it.
STEP 2 (Automatic)
Once the experimental spectrum is uploaded, apm2s automatically triggers peak picking and generates a list of experimental monoisotopic centroids. For fine-tuning of similarity calculations, the evolution of the resolution as a function of the m/z (which depends on each type of mass analyzer) and the corresponding regression curve are used to adjust the peak width. Both calculations can be viewed in the Peak Picking Module. The data showed in the left represent the resolution (peak width) vs m/z in our Orbitrap Elite for the MS/MS of ACTH 18-39 example available from List of experimental spectra.
STEP 3
As a next step, you should check the apm2s selector and define the main physical properties of the target peptide/small protein such as sequence (one or three letters code) where you can add the known modifications in brackets. The list of all abbreviations that can be used to indicate the common functional groups is found at the top right corner of the Settings window. Finally you need to specify the ionization method by indicating the expected adducts/charge carriers. Go to our glossary for more details.
STEP 4
All other suspected groups with unknown localization (such as metals, variable modifications, PTMs, small molecules adducts, etc…) can be additionally indicated in the Variable groups section, separated by a comma. You should end the list of groups by a comma (,) if you want to allow the test of absence of the listed groups (none of the possibilities). apm2s will generate the molecular formula for all possible combinations between the selected fragments of a given sequence with the additional groups (at different positions) for all selected ways of ionization.This option was not selected for any of our examples in the list of experimental spectra.
STEP 5
The user can filter the obtained molecular formula in step 5 by different criteria such as the charge, the experimental m/z or the unsaturation. This option was not selected for any of our examples in the list of experimental spectra.
STEP 6
In order to improve the isotopic profile matching between theoretical and experimental data a few spectrum filters should be properly adjusted here. First you should set the experimental relative peak intensity threshold (typically between 0.01-0.1). Second, the authorized experimental mass error in ppm (typically <5 for Orbitrap mass analyzer) and third, the minimum similarity for peak matching should be set (typically between 70-80%). Check how Apm2s calculates Similarity here. Lastly, you have to decide if the monoisotopic mass has to be present in the spectrum (which depends on the MW of the peptide/protein) and set the comparison zone which should be adjusted based on the complexity of the expected isotopic pattern.
STEP 7
For analysis of protein after enzymatic digestion, you should specify different parameters to run the calculation: the enzyme used for digestion (click on the arrow to see the different options), the number of possible missed cleavages and the minimum/maximum number of residues allowed per peptide. This option was not selected for any of our examples in the list of experimental spectra given.
STEP 8
Before running the apm2s calculation, you can select any of the six possible fragmentation sites along the protein backbone using the Roepstorff and Fohlman nomenclature defined here. If the charge is retained on the N terminal fragment, the ions are classified as either a-ion, b-ion or c-ion. When the C-terminal retains the charge, the ions are named x-ion, y-ion or z-ion. Internal cleavages Y/A and Y/B with a user-defined length can also be considered in Internal fragments section. Finally, Immonium ions and neutral losses can be also tracked. For all kind of fragmentation, it is possible to select all the cleavages at the same time clicking the checkmark square.
Step 9 (Optional)
Once the initial calculation is over, apm2s allows the user an additional post calibration step which could be performed to improve mass accuracy and, therefore, similarity scores of matched ions. In order to recalibrate the data, you need first to precise the minimum number of reference peaks with defined minimum similarity to be taken for the post calibration process within the Peak similarity parameters window. Once defined, you click the calibrate and recalculate button and apm2s reprocess all the data in a few seconds. You can view the graph of errors (in Da and in ppm) between found and expected after corrections and other details in the Peak picking and recalibration module at the top. Displayed graph of errors corresponds to our ACTH 18-39 example available from List of experimental spectra.
Option
Apm2s can also calculate all the possible theoretical fragmentations for any peptide & protein sequence as well as theoretical enzymatic digestion products without experimental data. For that, you can start the procedure in the step 3 by selecting Apm2s, type of peptide/protein, ionization and sequence. You can also add any given variable groups (Step 4) and use any of the molecular formula filters (Step 5). You can define the enzymatic digestion parameters (step 7) and ,finally select any or all the possible fragmentations (Step 8) and the software will generate automatically the theoretical cleavages within digestion products.
Resuts 1
The first view is a summarizing table named List of fragments. It contains all the information about the identified fragment ions. You can see the type of fragmentation, the molecular formula and its ionization form. You can see as well the Molecular formula monoisotopic peak (without ionization) and the final monoisotopic mass including the charge. Another interesting values such as the error (ppm), the relative abundance (related to the base peak), the quantity, the group and the similarity score are also included in this view. For any of the fragment ions, you can zoom and interact in the mass spectra view (Results 2). All the data presented in the table can be classified by any criterion with a simple click on the column header. You can also use the search bar in order to search faster an individual value or use it by range (go to glossary). You can also export the whole table as text file with Export Data button. The data showed in the left represent the MS/MS of ACTH 18-39 example available from List of experimental spectra.
Resuts 2
Mass spectra window displays the experimental data (in red) overlaid with the theoretical matched fragments (in blue). Identified fragment ions are assigned and color-coded on the top with details (in gray). You can select a single fragment in the table view (Results 1) and it will be highlighted in this view (in yellow). It is possible to zoom in (left button), zoom out (double left button) and show on the full screen (option bar at the top of the window) this window. The content of this window can be either printed or exported as SVG file (option bar). The ACTH 18-39 example after recalibration is shown in the figure (left).
Resuts 3
The Info box window shows details for the table-selected ion such as its name, monoisotopic mass, peptide composition, charge and ionization adduct. You’ll find as well the theoretical monoisotopic mass and the closest m/z experimental data. Below this window, the assigned quantity, the number of distinct fragments found and their percentage of the total intensity are shown. The total amount of theoretically possible fragments based on the peptidic sequence and the settings are displayed as well.
Resuts 4
The Similarity box window displays the matching of theoretical isotopic pattern to experimental spectrum for the selected fragment with differences in the intensity of isotopologues highlighted in yellow. You can check our concept of similarity here.
Resuts 5
The Graphic box window is displayed in the Report module. In this module, you will find the same list of fragments table where we can check/uncheck the desired fragment ions that will be included in the graphic view. In the example, we selected just the sequence fragment ions with a similarity >90% for ACTH 18-39 example. y-ion fragment ions are in red whereas b-ions are in blue. Internal fragments are indicated as green bars (not shown here). Each fragment ion is also annotated with its nomenclature, charge and similarity in grey (<90%) or black (>90%).
