Serum Degradation Analysis: what is it?

Biological samples undergoing long term cryopreservation are used in retrospective studies for biomarker identification. Moreover, the handling of samples before freezing may affect their quality. The quality of source material must be assessed before any study. The peptidomic profile of a serum sample is characterized by some fibrinopeptide related signals, mainly fibrinopeptide A (fpA) and its degradation products, generated during coagulation. The high susceptibility of fpA to degradation, which was observed in a set of samples (see a description of the samples and MS spectra data in the data page) both analysed within 2 hours from collection and after storage of 18 months at -80°C, suggests its use as a quality indicator for cryopreserved serum samples. When properly preserved, they should mainly contain whole and little degraded forms of fpA. Samples exposed to a greater extent to chemico-physical injuries should show a greater presence of shorter peptides.
We defined a mass spectrometry based method and developed a software tool to assess the integrity level of serum samples by evaluating their fibrinopeptide contents. The method also takes into account a serum sample of good quality, analysed with the same procedure, to be used as a reference.

Serum Degradation Analysis: how does it work?

The Serum Degradation Analysis software processes spectra to extract peak lists, which are then elaborated for noise reduction. Finally, fpA related peaks are compared in order to compute their overall abundance and the percent contribution of each peptide. Abundances are expressed as percent of the abundance of a reference spectrum. If a reference spectrum is not provided by the user, the spectrum with the greatest overall abundance for fpA peptides is taken as the reference. A quality score is assigned to spectra by taking into account both their overall fpA abundance and the ratio between abundances of more and less degraded forms. Elaboration is optimized by processing spectra in parallel on a multicore virtual server.

One interface, three forms

Serum Degradation (SeraDeg) software comes to you with three input forms. This is mainly due to the fact that your data need to be uploaded to the server and this can take some time, depending on its size and your internet connection. So, in order to avoid uploading many times the same data, you are allowed to upload it once and make reference to the uploaded file any time you need. Of course, we cannot store all your data forever. We presently allow storage for six months. This should be long enough to allow you all needed analyses. However, if your data has a limited size and/or you only need to analyse it once or a few times, you can upload and analyse it in a single step.

Uploading your data for repeated analysis

In order to upload your data for later analyses, go to the Upload form.
In this form, you must specify: i) a reference name for the data, ii) your email address, iii) the file you want to upload. See figure 1.
This information is used to make your upload unique and retrievable. When you will analyse this data, you will have to specify these three information.
The name of your country is only requested for statistical purposes, and you can disregard it (but please, insert it in the form).
See a description of the parameters and of the format of the file in the section on "Data formats and parameters" of this manual.

Figure 1: Upload form
Click to open enlarged in a new tab.

Starting an analysis on uploaded data

Once you have uploaded your data, you can analyse them by using the Analyse form.
In this form, you must specify the same key information defined while uploading the file: i) reference name, ii) email address, iii) file name. Moreover, you must specify a job name, which will be a unique identifier for the analysis, along with the execution parameters. See figure 2.
The name of your country is only requested for statistical purposes, and you can disregard it (but please, insert it in the form).
See a description of the parameters and of the format of the file in the section on "Data formats and parameters" of this manual.

Figure 2: Analyse form
Click to open enlarged in a new tab.

Uploading and analysing at once

Instead of uploading and analysing data in distinct steps, you can upload and analyse it at once by using the Upload and analyse at once form.
This is especially useful if you only want to analyse the data once (e.g., when you are confident on the parameters to use) or if the data file is small (hence the upload time is limited).
In this form, you must specify the job name, which will be a unique identifier for the analysis, the file to be uploaded and the execution parameters. See figure 3.
Your email address is required for returning some essential information on the analysis by email. You can omit it.
The name of your country is only requested for statistical purposes, and you can disregard it (but please, insert it in the form).
See a description of the parameters and of the format of the file in the section on "Data formats and parameters" of this manual.

Figure 3: Upload and analyse at once form
Click to open enlarged in a new tab.

Summary results

Ongoing elaboration

Summary of the analysis and legends

Results

• Spectrum label, together with a link to see the detailed graphical output
• Score, which must be interpreted at the light of the legend above
• High mass peptides percentage, which is computed by taking into account high and low mass peptides only
• Overall abundance of all fpA peptides for the given spectrum
• Overall abundance of all fpA peptides expressed in comparison to the overall abundance of the reference spectrum
• Deviation, which is the Euclidean distance between the spectrum and the reference computed by taking into account all fpA peptides
• Abundance of all fpA peptides in the given spectrum, in separate columns

Figure N: Summary results of the analysis
The results of the analysis are reported in a table where each line makes reference to a distinct spectrum. Lines are ordered by decreasing total fpA peptide abundance.
This figure only includes the upper part and two intermediate parts of the output. In the upper part, a summary of the analysis and the table legends are reported. In the following sections, two distinct situations are shown. In the first one, the lines where the quality score becomes orange are shown, while in the second section the passage from orange to red scores is shown.

Detailed results

A detailed output can be obtained for each spectrum by clicking on the "Graph" button of the summary output page. See the figure below.
In this detailed output, a few graphs and tables are used to show the distribution of fpA peptides in the spectrum. As shown in the following figure, the output is presented in two rows. In the upper row, from left to right, are reported:

• the job name, some essential data on the reference under analysis (label, overall abundance of fpA peptides and the ratio between low and high mass peptides), and a bullet graph for the overall fpA abundance in the spectrum compared to the reference spectrum (see Wikipedia for a definition of a bullet graph),
• a gauge graph representing the percent of high mass fpA peptides in the spectrum compared to the sum of both high and low mass peptides,
• a bar graph representing both low mass and high mass percentages.

• a bar graph with the distribution of fpA peptide abundances in percent, which includes all fpA related peptides,
• a table with the fpA peptide abundances, both absolute and percent.

Figure N: Detailed results for a single spectrum
The results of the analysis are reported in a table where each line makes reference to a distinct spectrum. Lines are ordered by decreasing total fpA peptide abundance.
This figure only includes the upper part and two intermediate parts of the output. In the upper part, a summary of the analysis and the table legends are reported. In the following sections, two distinct situations are shown. In the first one, the lines where the quality score becomes orange are shown, while in the second section the passage from orange to red scores is shown.

Information returned by email

When uploading an archive for later analysis

• The reference name for the upload.
• The email address of the user.
• The file name of the uploaded archive.

From: paolo.romano@hsanmartino.it [mailto:paolo.romano@hsanmartino.it] 
Sent: Wednesday, April 12, 2017 8:52 AM
To: Romano Paolo
Subject: SeraDeg upload results

The file testAllControls.zip has been uploaded.
It will remain available until Thursday 12 October 2017.
The following credentials have been recorded in association with this upload:

Reference name: seradeg_allControls.
User email: paolo.romano@hsanmartino.it.

At the end of an analysis

Input data format

Spectra

100.08097 2.49256
100.09243 14.5082
100.103 30.0608
100.11535 40.5548
......

Output data format

Intermediate data

• LABEL_spectrum.txt: a copy of the spectrum in input.
• LABEL_sp_MZ.txt: the values of the spectrum in a range of -2.3 - +2.3 m/z around the MZ peptide (10 files, one per peptide).
• LABEL_peaks.txt: the list of peaks detected by the local maxima criterion for the whole spectrum.
• LABEL_selectedPeaks.txt: the list of peaks selected by the median filter for the whole spectrum.
• LABEL_peptide_values.txt: the abundances identified for the 10 fpA peptides in the spectrum.
• LABEL_k_peptide_values.txt: the abundances identified for peaks having exactly -1 m/z with respect to the 10 fpA peptides in the spectrum.
• LABEL_sp_MZ.png: the figure of the portion of the spectrum corresponding to the data in the file LABEL_sp_MZ.txt (10 files, one per peptide).
• LABEL_sp_MZ_th.png: a reduced figure (thumbnail) of LABEL_sp_MZ.png for visualization in the detailed output (10 files, one per peptide).

Parameters