New Statistical Algorithms for the Analysis of Mass - FU Berlin, FB MI ...

4.1. DATA USED 67
4.1.2 Serum Data Used
The data was acquired by our partner organization the Institute of Laboratory
Medicine, Clinical Chemistry and Molecular Diagnostics at the University
Hospital Leipzig. The biological foundation consists of frozen serum samples of
770 apparently healthy blood donors who served as basis for the mass spectrometric
investigation. The study group has been described elsewhere (Kratzsch
et al., 2005).
Human serum is particularly susceptible to any confounding factor, for example,
(Boguski and McIntosh, 2003) showed that it does make a difference
whether the subject is sitting or recumbent during blood taking and can induce
a change by 10% in total protein concentrations. Further, the proteome profiles
can also be significantly influenced by the pre-analytical process such as
collection of samples (time-frames) and how the processing is done, e.g. variables
of clotting tube types, freeze-thaw cycles, centrifugation, storage time
and temperature, and so forth (Banks et al., 2005). Based on these results,
(Baumann et al., 2005) developed a protocol for preanalytics that was used in
this study.
Proteome fractionation: After thawing on ice peptide and protein purification
and fractionation was performed using a magnetic bead-based separation technique
(ClinProt TM , Bruker Daltonics, Leipzig, Germany) with specific surface
functionalities (MB-IMAC Cu, MB-WCX, MB-HIC C8).
Chemicals & Consumables: Gradient grade acetonitrile, ethanol, acetone were
obtained from J.T. Baker (Phillipsburg, USA); trifluoroacetic acid was purchased
from Sigma-Aldrich (Steinheim, Germany). Peptide and protein calibration
standards, a-cyano-4-hydroxycinnamic acid were purchased from Bruker
Daltonics (Leipzig, Germany). Peptide preparations were done in 0.2ml polypropylene
tubes (8-tube strips) from Biozym (Hess. Oldendorf, Germany). The
MALDI-TOF AnchorChip TM target (four spots) was purchased from Bruker
Daltonics (Leipzig, Germany). The protein calibration standard mix (Part
No.: 206355 & 206196) used for the spiking experiments was bought from
Bruker Daltronics (Leipzig, Germany).
Hardware Configuration: ClinProtTM Robot & Autoflex Linear MALDI-TOF
Mass Spectrometer (Bruker Daltonics, Germany)
Mass Spectrometry: Mass spectra were recorded by the flexControl TM 2.0 Software
(Bruker Daltonics, Germany). The settings were applied as follows: Ion
source 1: 20 kV; ion source 2, 18.50 kV; lens, 9.00 kV; pulsed ion extraction,
120 ns; nitrogen-pressure, 2500 mbar. Ionization was achieved by a nitrogen
laser (λ=337 nm) operating at 50 Hz. For matrix suppression a high gating
factor with signal suppression up to 500 Da was used. Mass spectra were detected
in linear positive mode. Spectral data were combined with beforehand
surveyed epidemiological and clinical metadata in a Microsoft SQL Server TM
database to provide highly differentiated classification criteria (such as age,
gender, or even peptide admixture).
Overall, about 8500 spectra were acquired during data collection. To cope
with the amounts of raw data and data generated during the analyses, and to
enable access to one structured storage (as opposed to many different files), a
Microsoft SQL Server TM database was used.