Effects of Traveling Wave Ion Mobility Separation on Data Independent Acquisition in Proteomics Studies

qTOF mass spectrometry and traveling wave ion mobility separation (TWIMS) hybrid instruments (q- TWIMS-TOF) have recently become commercially available. Ion mobility separation allows an additional dimension of precursor separation inside the instrument, without incurring an increase in instrument time. We comprehensively investigated the effects of TWIMS on data-independent acquisition on a Synapt G2 instrument. We observed that if fragmentation is performed post TWIMS, more accurate assignment of fragment ions to precursors is possible in data independent acquisition. This allows up to 60% higher proteome coverage and higher confidence of protein and peptide identifications. Moreover, the majority of peptides and proteins identified upon application of TWIMS span the lower intensity range of the proteome. It has also been demonstrated in several studies that employing IMS results in higher peak capacity of separation and consequently more accurate and precise quantitation of lower intensity precursor ions. We observe that employing TWIMS results in an attenuation of the detected ion current. We postulate that this effect is binary; sensitivity is reduced due to ion scattering during transfer into a high pressure “IMS zone”, sensitivity is reduced due to the saturation of detector digitizer as a result of the IMS concentration effect. This latter effect limits the useful linear range of quantitation, compromising quantitation accuracy of high intensity peptides. We demonstrate that the signal loss from detector saturation and transmission loss can be deconvoluted by investigation of the peptide isotopic envelope. We discuss the origin and extent of signal loss and suggest methods to minimize these effects on q-TWIMS-TOF instrument in the light of different experimental designs and other IMS/MS platforms described previously

the synapter

Optimising identification and quantitation by combining data usin

Improving Qualitative and Quantitative Performance for MS E -based Label-free Proteomics

Label-free quantitation by data independent methods (for instance MSE ) is growing in popularity due to the high technical reproducibility of mass spectrometry analysis. The recent introduction of Synapt hybrid instruments capable of incorporating ion mobility separation within mass spectrometry analysis now allows acquisition of high definition MSE data (HDMSE ). HDMSE enables deeper proteome coverage and more confident peptide identifications when compared to MSE , while the latter offers a higher dynamic range for quantitation. We have developed synapter as, a versatile tool to better evaluate the results of data independent acquisitions on Waters instruments. We demonstrate that synapter can be used to combine HDMSE and MSE data to achieve deeper proteome coverage delivered by HDMSE and more accurate quantitation for high intensity peptides, delivered by MSE . For users who prefer to run samples exclusively in one mode, synapter allows other useful functionality like false discovery rate estimation, filtering on peptide match type and mass error, and filling missing values. Our software integrates with existing tools, thus permitting us to easily combine peptide quantitation information into protein quantitation by a range of different approaches

Improving Qualitative and Quantitative Performance for MS^E‑based Label-free Proteomics

Label-free quantitation by data independent methods (for instance MS^E) is growing in popularity due to the high technical reproducibility of mass spectrometry analysis. The recent introduction of Synapt hybrid instruments capable of incorporating ion mobility separation within mass spectrometry analysis now allows acquisition of high definition MS^E data (HDMS^E). HDMS^E enables deeper proteome coverage and more confident peptide identifications when compared to MS^E, while the latter offers a higher dynamic range for quantitation. We have developed synapter as, a versatile tool to better evaluate the results of data independent acquisitions on Waters instruments. We demonstrate that synapter can be used to combine HDMS^E and MS^E data to achieve deeper proteome coverage delivered by HDMS^E and more accurate quantitation for high intensity peptides, delivered by MS^E. For users who prefer to run samples exclusively in one mode, synapter allows other useful functionality like false discovery rate estimation, filtering on peptide match type and mass error, and filling missing values. Our software integrates with existing tools, thus permitting us to easily combine peptide quantitation information into protein quantitation by a range of different approaches

Improving Qualitative and Quantitative Performance for MS^E‑based Label-free Proteomics

Label-free quantitation by data independent methods (for instance MS^E) is growing in popularity due to the high technical reproducibility of mass spectrometry analysis. The recent introduction of Synapt hybrid instruments capable of incorporating ion mobility separation within mass spectrometry analysis now allows acquisition of high definition MS^E data (HDMS^E). HDMS^E enables deeper proteome coverage and more confident peptide identifications when compared to MS^E, while the latter offers a higher dynamic range for quantitation. We have developed synapter as, a versatile tool to better evaluate the results of data independent acquisitions on Waters instruments. We demonstrate that synapter can be used to combine HDMS^E and MS^E data to achieve deeper proteome coverage delivered by HDMS^E and more accurate quantitation for high intensity peptides, delivered by MS^E. For users who prefer to run samples exclusively in one mode, synapter allows other useful functionality like false discovery rate estimation, filtering on peptide match type and mass error, and filling missing values. Our software integrates with existing tools, thus permitting us to easily combine peptide quantitation information into protein quantitation by a range of different approaches

Improving Qualitative and Quantitative Performance for MS^E‑based Label-free Proteomics

Label-free quantitation by data independent methods (for instance MS^E) is growing in popularity due to the high technical reproducibility of mass spectrometry analysis. The recent introduction of Synapt hybrid instruments capable of incorporating ion mobility separation within mass spectrometry analysis now allows acquisition of high definition MS^E data (HDMS^E). HDMS^E enables deeper proteome coverage and more confident peptide identifications when compared to MS^E, while the latter offers a higher dynamic range for quantitation. We have developed synapter as, a versatile tool to better evaluate the results of data independent acquisitions on Waters instruments. We demonstrate that synapter can be used to combine HDMS^E and MS^E data to achieve deeper proteome coverage delivered by HDMS^E and more accurate quantitation for high intensity peptides, delivered by MS^E. For users who prefer to run samples exclusively in one mode, synapter allows other useful functionality like false discovery rate estimation, filtering on peptide match type and mass error, and filling missing values. Our software integrates with existing tools, thus permitting us to easily combine peptide quantitation information into protein quantitation by a range of different approaches

Effects of Traveling Wave Ion Mobility Separation on Data Independent Acquisition in Proteomics Studies

qTOF mass spectrometry and traveling wave ion mobility separation (TWIMS) hybrid instruments (q-TWIMS-TOF) have recently become commercially available. Ion mobility separation allows an additional dimension of precursor separation inside the instrument, without incurring an increase in instrument time. We comprehensively investigated the effects of TWIMS on data-independent acquisition on a Synapt G2 instrument. We observed that if fragmentation is performed post TWIMS, more accurate assignment of fragment ions to precursors is possible in data independent acquisition. This allows up to 60% higher proteome coverage and higher confidence of protein and peptide identifications. Moreover, the majority of peptides and proteins identified upon application of TWIMS span the lower intensity range of the proteome. It has also been demonstrated in several studies that employing IMS results in higher peak capacity of separation and consequently more accurate and precise quantitation of lower intensity precursor ions. We observe that employing TWIMS results in an attenuation of the detected ion current. We postulate that this effect is binary; sensitivity is reduced due to ion scattering during transfer into a high pressure “IMS zone”, sensitivity is reduced due to the saturation of detector digitizer as a result of the IMS concentration effect. This latter effect limits the useful linear range of quantitation, compromising quantitation accuracy of high intensity peptides. We demonstrate that the signal loss from detector saturation and transmission loss can be deconvoluted by investigation of the peptide isotopic envelope. We discuss the origin and extent of signal loss and suggest methods to minimize these effects on q-TWIMS-TOF instrument in the light of different experimental designs and other IMS/MS platforms described previously