Application of SELDI-TOF mass spectrometry for the identification of differentially expressed proteins in transformed follicular lymphoma.
Completion of the human genome project has focused scientific attention on the development of methods that permit rapid characterization of proteins that are encoded by the genome. Recent improvements in two-dimensional separation techniques in combination with protein identification software/databases and mass spectrometry (MS) now permit rapid comprehensive large-scale analysis of individual proteins within complex protein mixtures. We have performed pairwise comparisons of low-grade and transformed follicular lymphomas (FLs) in order to identify proteins that may be involved in FL progression using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometer (ProteinChip, Ciphergen Biosystems). This system utilizes preactivated differential binding surfaces to achieve multidimensional chromatography. The protein-bound chips were then analyzed by a SELDI-TOF mass spectrometer to generate protein profiles. In preliminary experiments, we established that the MS data obtained from SELDI-TOF MS were reproducible, and that reduction in sample complexity improved the ability to detect lower abundance proteins. With specific regard to FL transformation, we rapidly identified a number of potential candidate proteins involved in this process. These included an upregulated 32 kDa protein and a down-regulated 11.8 kDa protein. Protein database searches revealed several candidates, among them cyclin D3 (32.5 kDa) and caspase 3 (11.8 kDa) whose differential expression were confirmed by immunoblotting and/or immunohistochemical analysis on the primary tissue specimens. Our studies demonstrate the utility of SELDI-TOF-MS for the rapid discovery of differentially expressed proteins using femtomolar quantities of crude protein derived from biopsy material. The versatility of this methodology supports its application to the rapid discovery of potential biomarkers in a variety of cellular systems.
Lin
Zhaosheng
Z
ARUP Institute for Clinical and Experimental Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.
Jenson
Stephen D
SD
Lim
Megan S
MS
Elenitoba-Johnson
Kojo S J
KS
D018951:Antigens, CD20 / Q000032:analysis; D015153:Blotting, Western; D017252:CD3 Complex / Q000032:analysis; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D056743:Cyclin D3; D016213:Cyclins / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007150:Immunohistochemistry; D008224:Lymphoma, Follicular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051560:Proto-Oncogene Proteins c-bcl-6; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014157:Transcription Factors / Q000032:analysis
D018951:Antigens, CD20; C534696:CCND3 protein, human; D017252:CD3 Complex; D056743:Cyclin D3; D016213:Cyclins; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051560:Proto-Oncogene Proteins c-bcl-6; D014157:Transcription Factors; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases