Targeted and untargeted metabolic profiling by incorporating scanning FAIMS into LC-MS Kayleigh Arthur K.Arthur@lboro.ac.uk
Introduction LC-MS is a highly used technique for untargeted profiling analyses Incorporation of scanning ion mobility with LC-MS Why choose FAIMS over IMS (DT or TW)? Fast FAIMS scanning achievable with miniaturised FAIMS device Full scan FAIMS within time of a UHPLC peak Approach applicable to a range of mass spectrometers
FAIMS
FAIMS
FAIMS To MS
Untargeted profiling IMS Harry et al, J. Chromatogr. B, 28, 871, 357-361 Valentine et al, J. Proteome. Res., 26, 5, 2977-2984
Untargeted profiling FAIMS No LC LC run time = 12 mins FAIMS scan time = 2-3 s Beach et al, Anal. Chem., 211, 83, 917-9113 Canterbury et al, Anal Chem, 28, 8, 6888-6897 Creese et al, J Am Soc Mass Spectrom, 213, 24, 431-443 ETD Scanning can miss ions if the top of the LC peak does not coincide with CV for transmission 6 FAIMS conditions Peptide IDs missed with scanning mode CID
FAIMS-MS TWIMS-MS FAIMS vs TWIMS Direct comparison FAIMS covers a greater proportion of the analytical space FAIMS covers across the CF range at all m/z TWIMS shows a correlation between m/z and bin number o Bin number increases as m/z increases
Apply approach to human urine Incorporate into Omics workflow Set-up Modes of operation Acquisition of nested data sets
Miniaturised FAIMS with LC-MS
How? FAIMS/LC-MS Synchronisation Computer Contact closure interface Contact closure board fitted to LC binary pump Agilent 623 TOF MS Owlstone Miniaturised FAIMS chip ESI Source Agilent 12 series LC FAIMS control unit
LC-FAIMS-MS Modes of Operation Static Scanning Fixed Dispersion Field Fixed Dispersion Field Fixed Compensation Field Scanning Compensation Field on chromatographic peak timescale
Application to Biological Matrices FAIMS-MS Optimisation of FAIMS DF and CF Targeted LC-FAIMS-MS Isobaric separation, reduction in chemical noise, in-source CID Untargeted LC-FAIMS-MS Feature determination
Scanning LC-FAIMS-MS how? Chromatographic Peak LC peak width Compensation Field Scan 11 CFs per s Mass Spectra 1 per CF All CFs scanning
DF and CF Selection for Untargeted Analysis of Human Urine CF resolution Intensity FAIMS-MS
Human Urine TIC Scanning approach 6 x1 2. 1.5 1..5..5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. Retention Time (min) x1 5 7 6 5 4 3 2 1 6.8 7 7.2 7.4 7.6 7.8 8 8.2 Counts vs. Retention Time (min)
Human Urine TIC Scanning approach CF deconvolution into individual channels CF adds another dimension of separation
Peak Area Peak Area Urinary Creatinine 6 Urine 6 Urine 5 Standards 5 Standards 4 4 3 3 2 2 1 1-1 1 2 3 4-1 1 2 3 4 Compensation Field (Td) Compensation Field (Td) [M+H] + m/z 114.66 [M+Na] + m/z 136.48
LC Peak Area Isobaric Separation 2 15 1 5-1 1 2 3 4 m/z 27.11 RT 5.21 min 4 x1 2 1 5.212 LC-MS FAIMS off FAIMS Compensation Field (Td) 3 x1 5.217 3 x1 3 LC-FAIMS-MS 2 1 LC-FAIMS-MS CF.8 Td 2 1 5.1 5.2 5.3 Counts vs. Retention Time (min) FAIMS off = 1 feature FAIMS on = 2 features x1 3 1.5 5.23 LC-FAIMS-MS CF 2.4 Td 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 Counts vs. Retention Time (min)
Reduction in Chemical Noise and Interferences m/z 331.21, RT 4.24 min FAIMS off S:N = 2.7 FAIMS on S:N = 124.1 3 x1 4 2 x1 3 2 1 LC-MS FAIMS off LC-FAIMS-MS CF.8 Td 1 2 3 4 5 6 7 8 9 Counts vs. Retention Time (min) FAIMS off = features, FAIMS on = 1 features
Frag 35 V Frag 35 V Frag 2 V Frag 2 V In-source CID vs FAIMS-selected CID LC-MS LC-FAIMS-MS x1 4 2 Precursor ion 181.7 26158 x1 2 5 Precursor ion 181.7 534 95.6 173 121.4 435 141.7 3514 169.97 1489 x1 3 4 2? 91.5 1225? 15.5 1614? 124.5 2279 141.7 893 181.8 437 x1 2 4 2 124.5 18 Fragment 181.8 468 1 12 14 16 18 Counts vs. Mass-to-Charge (m/z) 1 12 14 16 18 Counts vs. Mass-to-Charge (m/z) FAIMS-IN-SOURCE COLLISION INDUCED DISSOCIATION FISCID
Untargeted Feature Determination RT m/z Identified Feature RT DF + CF m/z Identified Feature
Acquisition of Nested Data Sets Identified features based upon RT, m/z and CF
Conclusions Acquisition of LC-FAIMS-MS nested data sets on timescale of UHPLC peak for the first time Increase in peak capacity using LC-FAIMS-MS in comparison to LC-MS Higher level of orthogonality with m/z / RT than IMS Separation of isobaric and co-eluting analytes DF + CF as additional identifiers Can be integrated into non-targeted omics workflows Applicable to a range of mass spectrometers
Acknowledgments Loughborough University: Colin Creaser James Reynolds Matthew Turner Owlstone: Lauren Brown Staff and researchers at the Centre for Analytical Science Contact me: K.Arthur@lboro.ac.uk