Ultrasonic assisted protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sonoreactor versus ultrasonic probe.
Two different ultrasonic energy sources, the sonoreactor and the ultrasonic probe, are compared for enzymatic digestion of proteins for protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the peptide mass fingerprint (PMF) procedure. Variables such as (i) trypsin/protein ratio; (ii) sonication time; (iii) ultrasound amplitude; and (iv) protein concentration are studied and compared. As a general rule, the trypsin/protein ratio and the minimum protein concentration successfully digested are similar with both ultrasonic energy sources. Results showed that the time needed to digest proteins was shorter with the ultrasonic probe, 60s versus 120s, for the same amplitude of sonication, 50%. However, lower standard deviations and cleaner MALDI-TOF-MS spectra were obtained with the sonoreactor. In addition, the sonoreactor device provided higher sample throughput (6 samples for the sonoreactor versus 1 sample for the ultrasonic probe) and easier sample handling for lower sample volumes (25 microl). Finally, a comparison of both methodologies for the specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the procedure.
Rial-Otero
R
R
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal.
Carreira
R J
RJ
Cordeiro
F M
FM
Moro
A J
AJ
Santos
H M
HM
Vale
G
G
Moura
I
I
Capelo
J L
JL
D045285:Desulfovibrio desulfuricans / Q000201:enzymology; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods; D021122:Protein Subunits / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry*; D013010:Sonication / Q000295:instrumentation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D014465:Ultrasonics
D021122:Protein Subunits; D011506:Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D014357:Trypsin