Differential capture of serum proteins for expression profiling and biomarker discovery in pre- and posttreatment head and neck cancer samples.
BACKGROUND
A long-term goal of our group is to develop proteomic-based approaches to the detection and use of protein biomarkers for improvement in diagnosis, prognosis, and tailoring of treatment for head and neck squamous cell cancer (HNSCC). We have previously demonstrated that protein expression profiling of serum can identify multiple protein biomarker events that can serve as molecular fingerprints for the assessment of HNSCC disease state and prognosis.


METHODS
An automated Bruker Daltonics (Billerica, MA) ClinProt matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer was used. Magnetic chemical affinity beads were used to differentially capture serum proteins prior to MALDI-TOF analysis. The resulting spectra were analyzed using postprocessing software and a pattern recognition genetic algorithm (ClinProt 2.0). An HNSCC cohort of 48 sera samples from 24 patients consisting of matched pretreatment and 6 to 12 month posttreatment samples was used for further analysis. Low-mass differentially expressed peptides were identified using MALDI-TOF/TOF.


RESULTS
In the working mass range of 1,000 to 10,000 m/z, approximately 200 peaks were resolved for ionic bead capture approaches. For spectra generated from weak cation bead capture, a k-nearest neighbor genetic algorithm was able to correctly classify 94% normal from pretreatment HNSCC samples, 80% of pretreatment from posttreatment samples, and 87% of normal from posttreatment samples. These peptides were then analyzed by MALDI-TOF/TOF mass spectometry for sequence identification directly from serum processed with the same magnetic bead chemistry or alternatively after gel electrophoresis separation of the captured proteins. We were able to compare this with similar studies using surface-enhanced laser desorption ionization (SELDI)-TOF to show this method as a valid tool for this process with some improvement in the identification of our groups.


CONCLUSIONS
This initial study using new high-resolution MALDI-TOF mass spectrometry coupled with bead fractionation is suitable for automated protein profiling and has the capability to simultaneously identify potential biomarker proteins for HNSCC. In addition, we were able to show improvement with the MALDI-TOF in identifying groups with HNSCC when compared with our prior data using SELDI-TOF. Using this MALDI-TOF technology as a discovery platform, we anticipate generating biomarker panels for use in more accurate prediction of prognosis and treatment efficacies for HNSCC.
Department of Microbiology and Molecular Cell Biology, Center for Biomedical Proteomics, Eastern Virginia Medical School, 700 W. Olney Road, Norfolk, VA 23507, U.S.A.
Gary L
GL
Freed
Lisa H
LH
Cazares
Craig E
CE
Fichandler
Thomas W
TW
Fuller
Christopher A
CA
Sawyer
Brendan C
BC
Stack
Scott
S
Schraff
O John
OJ
Semmes
J Trad
JT
Wadsworth
Richard R
RR
Drake
D000345:Affinity Labels; D000368:Aged; D000369:Aged, 80 and over; D000465:Algorithms; D015415:Biomarkers / Q000097:blood; D014408:Biomarkers, Tumor / Q000097:blood*; D001798:Blood Proteins / Q000032:analysis*; D002294:Carcinoma, Squamous Cell / Q000097:blood / Q000628:therapy*; D016022:Case-Control Studies; D015331:Cohort Studies; D005260:Female; D005500:Follow-Up Studies; D020869:Gene Expression Profiling / Q000379:methods*; D006258:Head and Neck Neoplasms / Q000097:blood / Q000628:therapy*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010363:Pattern Recognition, Automated; D011379:Prognosis; D011446:Prospective Studies; D040081:Protein Array Analysis; D012907:Smoking / Q000097:blood; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D015415:Biomarkers; D014408:Biomarkers, Tumor; D001798:Blood Proteins