Serum Degradation Analysis
Build August 10, 2017.
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TitleA mass spectrometry based method and a software tool to assess degradation status of serum samples to be used in proteomics for biomarker discovery.
We investigated the gradual fibrinopeptide A degradation as a result of the serum preservation process and proposed the fpA degradation pattern as a biomarker for the evaluation of sera integrity. As a consequence, we have developed SeraDeg, an original software able to automate the whole data analysis process.
This work has been submitted to the Journal of Proteomics and it is currently under review.
In this page, you will find links to the MS spectra which were the basis of our proposal. Specra are formatted according to the mzML standard.
Sample Processing Protocol
Serum samples from healthy donors were analyzed within two hours from collection and after a storage of 18 months at -80°C. All samples were analyzed by AP-MALDI/ToF MS after extraction of low molecular weight protein fraction by means of C18 magnetic beads. Each sample was analyzed in quadruplicate in reflectron positive mode on a 6210 ToF liquid chromatography (LC)/MS instrument (Agilent, Santa Clara, CA, USA) coupled with an atmospheric pressure pulsed dynamic focusing (PDF)-MALDI ion source (Agilent) equipped with a 337-nm nitrogen laser. Data acquisition was automated through Mass Hunter software (Agilent) and we decided to accumulate 600 shots per spectrum. The irradiation program was automated using the spiral motion control of the PDF-MALDI ion source. The first replicate, out of the programmed four, was discarded in order to avoid cross-contamination between consecutive samples.
Data Processing Protocol
Data handling was performed using PROTEO, a bioinformatic tool that allows both the bundling of isotopic abundances assigned to a single peptide and the normalization of the abundance of each m/z signal against the abundance of a synthetic peptide used as an internal standard (monoisotopic = 1,419.76 Da). Furthermore, PROTEO allows the selection of signals above a modulated abundance threshold built on the mass spectra profile.
A second bioinformatic tool, NEAPOLIS, was used both to obtain the mean abundance values for triplicate samples and to align mass spectra along the m/z axis, thereby facilitating subsequent export of the data as a matrix of m/z values and relative abundances.
These two tools have been incorporated in the Geena 2 tool.
ContactsAldo Profumo (Ospedale Policlinico San Martino - Genoa, Italy)
Paolo Romano (Ospedale Policlinico San Martino - Genoa, Italy)
AnnotationSpecies: Homo sapiens (Human)
Instrument: 6210 ToF liquid chromatography (LC)/MS instrument (Agilent, Santa Clara, CA, USA) coupled with an atmospheric pressure pulsed dynamic focusing (PDF)-MALDI ion source.
Software: PROTEO, NEAPOLIS, Geena 2.
Modification: Not available
Quantification: Not available
Experiment Type: Peptidome profiling
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