Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics.
We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.
Tomazela
Daniela M
DM
Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065, USA.
Patterson
Bruce W
BW
Hanson
Elizabeth
E
Spence
Kimberly L
KL
Kanion
Tiffany B
TB
Salinger
David H
DH
Vicini
Paolo
P
Barret
Hugh
H
Heins
Hillary B
HB
Cole
F Sessions
FS
Hamvas
Aaron
A
MacCoss
Michael J
MJ
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D040901:Proteomics / Q000191:economics / Q000379:methods*; D037701:Pulmonary Surfactant-Associated Protein B / Q000032:analysis*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D014132:Trachea / Q000737:chemistry*
D037701:Pulmonary Surfactant-Associated Protein B