Approximately 1.2 μg of glycopeptide mixture was reconstituted in 4 μl of nanopure water and further separated using a 30-cm-long, 75-μm-i.d. Hypercarb (porous graphitic carbon) column (Thermo Scientific, San Jose, CA, USA) and a nanoACQUITY UltraPerformance LC system (Waters, Manchester, UK). The mobile phases were the same as for ZIC–HILIC enrichment described above except that mobile phases A and B were switched. The glycopeptides were eluted using a gradient from 5 to 50% mobile phase B (0.1% FA in ACN) over 58 min at 400 nl/min directly into a Thermo LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The LTQ Orbitrap Velos was operated in a data-dependent MS/MS mode with the top eight ions (intensity) per duty cycle selected for HCD. In the initial experiments, a series of four NCEs (30, 40, 50, and 60%), for HCD were tested to determine optimal collisional energy (i.e., ideal HCD spectra). In the following experiments, the LTQ Orbitrap Velos was also operated in a data-dependent mode with the top eight ions and the HCD acquisitions automatically switching between NCEs of 30 and 50%.
Thermo ltq orbitrap velos mass spectrometer
Manufactured by Thermo Fisher Scientific
The Thermo LTQ Orbitrap Velos mass spectrometer is a high-performance analytical instrument designed for advanced proteomics and metabolomics research. It combines the linear ion trap (LTQ) technology with the Orbitrap mass analyzer, providing high-resolution, accurate mass measurements for the identification and quantification of complex molecular samples.
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3 protocols using thermo ltq orbitrap velos mass spectrometer
1
Glycopeptide Separation and Mass Spectrometry
2
Protein Purification and Mass Spectrometry
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Protein-affinity purification was performed as we have reported previously (Li et al, 2015 (link)). In brief, 2 liters of yeast cells were induced into synchronous meiosis for 6 h. Yeast cells were harvested and ground into powder in the presence of liquid nitrogen. The yeast powder was then stored at −80°C before use. For affinity purification, yeast powder was thawed in the extraction buffer. The lysate was then incubated with epoxy-activated M-270 Dynabeads (Cat. no. 14305D; Thermo Fisher Scientific), which were cross-linked with rabbit IgG (Cat. no. I5006; Sigma-Aldrich). The final product was eluted from the beads and dried for further study.
Purified protein samples were digested by trypsin. The proteomics work was carried out by the Translational Science Laboratory, Florida State University College of Medicine. An externally calibrated Thermo LTQ Orbitrap Velos mass spectrometer was used for mass spectrometry as per the method described previously (Li et al, 2015 (link)).
Purified protein samples were digested by trypsin. The proteomics work was carried out by the Translational Science Laboratory, Florida State University College of Medicine. An externally calibrated Thermo LTQ Orbitrap Velos mass spectrometer was used for mass spectrometry as per the method described previously (Li et al, 2015 (link)).
3
Protein Affinity Purification and Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein affinity purification was performed as we have reported previously (Li et al., 2015) . In brief, 2 liters of yeast cells were induced into synchronous meiosis for 6 hours. Yeast cells were harvested and ground into powder in the presence of liquid nitrogen. The yeast powder was then stored at -80°C before use. For affinity purification, yeast powder was thawed in the extraction buffer. The lysate was then incubated with epoxy-activated M-270 Dynabeads (Thermo Fisher Scientific, Cat#14305D), which were cross-linked with rabbit IgG (Sigma-Aldrich, Cat#I5006). The final product was eluted from the beads and dried for further study.
Purified protein samples were digested by trypsin. The proteomics work was carried out by the Translational Science Laboratory, Florida State University College of Medicine. An externally calibrated Thermo LTQ Orbitrap Velos mass spectrometer was used for mass spectrometry per the method described previously (Li et al., 2015) .
Purified protein samples were digested by trypsin. The proteomics work was carried out by the Translational Science Laboratory, Florida State University College of Medicine. An externally calibrated Thermo LTQ Orbitrap Velos mass spectrometer was used for mass spectrometry per the method described previously (Li et al., 2015) .
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