Cryoconservation of Peptide Extracts from Trypsin Digestion of Proteins for Proteomic Analysis in a Hospital Biobank Facility

We
tested a semiautomated protocol for the proper storage and conservation
in a hospital biobank of tryptic peptide extracts coming from samples
with low and high protein complexity for subsequent mass spectrometry
analysis. Low-complexity samples (serum albumin, serotransferrin.
and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected
to standard in-gel trypsin digestion. For LC–MALDI–TOF/TOF
analysis, purified β-galactosidase and human serum samples were
in-solution digested following standard procedures and desalted with
C18 stage-tips. In both cases, peptides extracts were aliquoted in
individually 2D coded tubes, vacuum-dried, barcode-read, and stored
in an automated −20 °C freezer in the Biobank facility.
Samples were kept dried at −20 °C until the corresponding
time-point of analysis, then reconstituted in the proper buffer and
analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS)
or LC–MALDI-TOF/TOF following a highly reproducible pattern
to ensure the reproducibility of the results. Protein identification
was done with either Mascot or Protein Pilot as search engines using
constant parameters. Over a period of 1 year we checked six different
time points at days 0, 7, 30, 90, 180, and 365. We compared MS and
MS/MS protein score, number of identified peptides, and coverage of
the identified proteins. In the low complexity samples, the number
of peptides detected gradually decreased over time, especially affecting
the MS score. However, two of the three proteins – serum albumin
and serotransferrin – were identified by both PMF and MS/MS
at day 90. By day 180, only MS/MS identification in some replicates
was possible. By LC–MS/MS, β-galactosidase and the most
abundant serum proteins were identified with good scores at all time
points even by day 365, with no detectable peptide loss or decrease
in the fragmentation efficiency, although a progressive decrease in
peptide intensity indicates that detection of low abundant proteins
could not be optimal after very long periods of time. Our results
encourage us to use the biobank facility in the future for long-term
storage – up to 3 months – of dried peptide extracts.