Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.
OBJECTIVE
Limited research to date has characterized the potential for HRPO to function as a primary molecular probe.


METHODS
Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry.


RESULTS
Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments.


CONCLUSIONS
HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.
Coyne
Cody P
CP
Department of Basic Sciences, College of Veterinary Medicine at Wise Center, Mississippi State University, Mississippi State, Mississippi 39762, USA. coyne@cvm.msstate.edu
Rashmir-Raven
Ann
A
Jones
Toni
T
Mochal
Cathleen
C
Linford
Robert L
RL
Brashier
Michael
M
Eddy
Alison
A
D000818:Animals; D026481:Blotting, Far-Western; D001826:Body Fluids / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000378:metabolism; D006736:Horses; D008168:Lung / Q000737:chemistry*; D037601:Mannose-Binding Lectin / Q000737:chemistry*; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D015336:Molecular Probe Techniques; D011485:Protein Binding; D011506:Proteins / Q000032:analysis
D037601:Mannose-Binding Lectin; D008567:Membranes, Artificial; D011506:Proteins; D006735:Horseradish Peroxidase