Ultra performance liquid chromatography isotope dilution tandem mass spectrometry for the absolute quantification of proteins and peptides.
A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.
Luna
Leah G
LG
Division of Laboratory Science, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, Georgia 30341, USA.
Williams
Tracie L
TL
Pirkle
James L
JL
Barr
John R
JR
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000276:immunology; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry / Q000276:immunology; D007252:Influenza Vaccines / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D011865:Radioisotope Dilution Technique; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology
D007252:Influenza Vaccines; D010455:Peptides; D011506:Proteins; D014764:Viral Proteins