On-line digestion system for protein characterization and proteome analysis.
An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research (rapid screening of protein-based drugs).
López-Ferrer
Daniel
D
Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Petritis
Konstantinos
K
Lourette
Natacha M
NM
Clowers
Brian
B
Hixson
Kim K
KK
Heibeck
Tyler
T
Prior
David C
DC
Pasa-Tolić
Ljiljana
L
Camp
David G
DG
Belov
Mikhail E
ME
Smith
Richard D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000191:economics / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011312:Pressure; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D012709:Serum Albumin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
D001426:Bacterial Proteins; D011506:Proteins; D012709:Serum Albumin; D014357:Trypsin