Global Phosphoproteome Analysis Using High-Field Asymmetric Waveform Ion Mobility Spectrometry on a Hybrid Orbitrap Mass Spectrometer.
Anal Chem
; 92(24): 15959-15967, 2020 12 15.
Article
en En
| MEDLINE
| ID: mdl-33270415
ABSTRACT
Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15-20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Fosfoproteínas
/
Espectrometría de Masas
/
Proteómica
Idioma:
En
Revista:
Anal Chem
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos