Assessment of buffer systems for harvesting proteins from tissue interstitial fluid for proteomic analysis.
Tissue interstitial fluid (TIF) bathes cells in tissues, and it is hypothesized that TIF proximal to a developing tumor may contain an enriched population of tumor-specific shed and secreted proteins relative to peripheral blood. Extraction of TIF proteins is typically accomplished through passive incubation of surgically resected tissues in phosphate buffered saline (PBS); however, its influence on cellular activity and viability has not been fully explored. The present investigation sought to characterize whether different buffer systems influence the recovered TIF proteome. Five TIF buffer systems were investigated including PBS, Dulbecco's modified Eagle medium (DMEM), and three organ transplantation preservative solutions: Celsior solution S (CS), histidine-tryptophan-ketoglutarate (HTK), and University of Wisconsin (UW). Kidney tumor, adjacent normal kidney, and ovarian tumor tissues were incubated in each of the buffer systems, and the harvested TIF proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the present results indicate that no significant differences exist in the recovered proteins from these two neoplasms between the five solution groups, additional sample preparative steps are required prior to LC-MS/MS for TIF proteins harvested from DMEM, UW, CS, and HTK. These data support that PBS is a suitable and convenient solution for harvesting TIF proteins for MS-based proteomics.
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, USA.
Pang-ning
PN
Teng
Bunja J
BJ
Rungruang
Brian L
BL
Hood
Mai
M
Sun
Melanie S
MS
Flint
Nicholas W
NW
Bateman
Rajiv
R
Dhir
Rohit
R
Bhargava
Scott D
SD
Richard
Robert P
RP
Edwards
Thomas P
TP
Conrads
D002021:Buffers; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002853:Chromatography, Liquid; D003470:Culture Media / Q000032:analysis; D004586:Electrophoresis; D045604:Extracellular Fluid / Q000737:chemistry*; D005260:Female; D006801:Humans; D007680:Kidney Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D019188:Organ Preservation Solutions; D010051:Ovarian Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012965:Sodium Chloride; D053719:Tandem Mass Spectrometry
D002021:Buffers; D003470:Culture Media; D019188:Organ Preservation Solutions; D011506:Proteins; D012965:Sodium Chloride