Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry.
Author(s): Villanueva J, Philip J, Entenberg D, Chaparro CA, Tanwar MK, Holland EC, Tempst P
Publication: Anal Chem, 2004, Vol. 76, Page 1560-70
PubMed ID: 15018552 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of specimen preparation method and MALDI-TOF protocols on serum peptide profiles. Serum was stored at -80 degrees C until analysis.
Summary of Findings:
Using only magnetic beads derivatized with C8 resulted in the largest number of MALDI-TOF peaks both in the 0.8-4Kda and 4-15 Kda range, and the authors report using beads with a porous surface improved binding capacity. The maximum number of peaks attainable was similar for all serum amounts, and a ratio of 50 ug beads/uL serum resulted in the highest number of MALDI-TOF peaks. More peaks resulted from elution with 50% MeCN at 1 uL MeCN per 500 ug beads than 20% or 70% MeCN, methanol, 2-propanol, or ethanol of from more or less MeCN, but two-step elution (20% MeCN, then 70% MeCN) increased the number of peaks by 8%. Pretreatment of serum with urea, urea/dithiothreitol (DTT), ethanol precipitation, cibachron blue, or ultrafiltration, with or without urea, decreased the number of MALDI-TOF peaks, and prewashing the serum tubes with n-octylglucoside did not increase the number of peptide peaks. Applying the serum to SCAX and eluting with increasing concentrations of sodium chloride applied to C8 beads increased the number of peaks by 30. Using HCCA as the MALDI-TOF matrix and 40% MeCN/50% methanol/10% water as the solvent resulted in more peaks than using 2,5-dihydroxybenzoic acid (DHB) or sinapic acid as the matrix and other concentrations of MeCN, methanol, 2-propanol, water and trifluoroacetic acid (TFA) as the solvent. Using 20-30 higher power laser shots prior to 100 laser shots at lower power increased the number of peaks compared to acquiring data with 100 lower power laser shots. Using the linear mode during acquisition rather than reflectron increased the number of peaks by a factor of 4. Similar spectra were observed when specimen handling was manual or automated. Use of the automated protocol allowed for identification of peptides differentially expressed between healthy controls and glioblastoma patients.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Normal volunteer
Glioblastoma
Biospecimen Aliquots and Components Filtration Centricon 30 ultrafiltration after incubation with urea
No filtration
Analyte Extraction and Purification Analyte purification Methanol
2-propanol
Ethanol
Acetonitrile
20%
50%
70%
1-step
2-step
C1 magnetic beads
C2 magnetic beads
C3 magnetic beads
C8 magnetic beads
C18 magnetic beads
C3 and C8 magnetic beads
C8 and C18 magnetic beads
C1 or C2 and then C8 magnetic beads
Various concetrations beads
Automated preparation
Biospecimen Acquisition Type of collection container/solution Untreated serum tube
Serum tube treated with n-octylglucoside
MALDI-TOF MS Specific Template modification Urea
Urea/DTT
Ethanol precipitation
Ultrafiltration
Cibachron blue
None
MALDI-TOF MS Specific Reaction solution Various concentrations of MeCN, methanol, 2-propanol, water and TFA
MALDI-TOF MS Specific Detection method 20-30 higher power laser shots prior 100 standard power shots
Standard laser power for all shots
MALDI-TOF MS Specific Technology platform Linear mode
Reflectron
Biospecimen Aliquots and Components Aliquot size/volume 5 ul serum
20 ul serum
50 ul serum
100 ul serum