Protein Identification (single gel band, LC-ESI-MS/MS)
Samples are digested with trypsin and the resulting peptides are analyzed by LC-ESI-MS/MS via the Orbitrap Velos Pro Mass Spectrometer and fragmented with CID fragmentation. Other types of fragmentation are available (HCD and ETD).
The results are searched against protein databases using the SEQUEST and/or MASCOT algorithms, and then the results are score filtered through Scaffold 3.0. Results are provided in the form of a Scaffold output file. You can download the Scaffold viewer here.
Protein Profiling of Complex Mixtures
(isolated fractions, IP’s, whole lane analysis)
We routinely analyze complex mixtures from a variety of preparations and isolations. A short conversation prior to sample submission usually is very helpful. The price of this analysis depends on the experimental design and the type of LC-MS/MS analysis performed.
Glycosylation analysis is the main focus of our research. We use a variety of sample preparations, LC-MS/MS analyses, and different software tools to verify the type and site of glycosylation. A pre-submission meeting or phone call is required to discuss the samples and desired analyses.
Glycan site localization is done by use of a combination of ion fragmentation techniques including ETD and HCD or CID. Usually through a series of 3-4 LC-MS/MS analyses the site can be localized [1-3 sites of glycosylation $500/sample; >3 sites $750/sample].
Our glycan screening on complex samples is done with intact glycopeptides and not released glycans. Usually samples have been probed with lectins or at least a glycoprotein staining kit before we undertake the analysis. Costs are dependent on the complexity of the sample.
Site-Specific Glycan Heterogeneity
Understanding the range of glycosylation chains present at a single site is a more involved process. Through a similar combination of LC-MS/MS analyses as we use for site localization plus a more extensive bioinformatic process we identify and verify the variable glycans observed at a single site. ($2000-$5000 per site depending on the complexity of the analysis, external starts at $5000).
Our experience with glycosylation analysis applies to many other forms of Post Translastional Modifications, and we have performed anlysis on a variety of modifications such as phosphorylation, oxidative modifications, and others. We recommend before submitting a sample that you set up a meeting with us to discuss the type of analysis that will be performed. (pricing same as for glycans).
LC-MS/MS results are typically searched by use of the SEQUEST and/or MASCOT search algorithms and then score filtered through Scaffold 3.0. We search the spectra against available protein databases or against one that you provide for us.
In addition to the standard search algorithms, we make use of Byonic Software (Protein Metrics) to search for PTMs. This software has been adapted for glycopeptide and glycan analysis which allows us to search for an unlimited amount of modifications.
Sample Preparation Price Per Sample
All samples that contain complex backgrounds (i.e. detergent, non-volatile salts, glycerol) should be submitted on a gel. A picture is required with the submission to make sure the appropriate bands are excised and digested. If you would like to use in-solution digestion, please contact us to discuss your sample.
All sample information regarding status and results can be found on our LIMS system. UAB Blazer id and password are required to enter this system. If you are not affiliated with UAB a username and password will be provided to you.
We strongly encourage you to contact us before submitting a sample, especially if submitting outside of UAB.
The majority of our service analysis takes the form of academic collaborations within and outside of UAB. This work reflects the mutual involvement of our laboratory and collaborators to make novel biomedical discoveries that involve the use of mass spectrometry and usually lead to publication and/or submission of grant proposals. In this case, there is still a minimum per day charge for the cost of running and maintaining the instrument.
For all our analysis, we have a series of quality control standards which we must meet before moving forward with our submitted sample analysis. This primarily falls under the categories of MS maintenance, LC-MS column conditioning and QC monitoring, and protein quantification of loaded samples to ensure we are performing the best possible analysis for those who entrust us with their samples.
Our laboratory provides a smaller range of services than typically seen for mass spectrometry service labs. That is because we are primarily a research laboratory as part of the UAB Stem Cell Institute in the Department of Biochemistry and Molecular Genetics. We do have a formalized service component in partnership with the UAB Cancer Center Mass Spectrometry/Proteomics (MSP) Shared Facility and the larger UAB Mass Spectrometry/Proteomics consortium. Our pricing, sample submission, data processing, data storage (LIMS), and billing all mirror and take advantage of the MSP Shared Facility infrastructure and organization. A full list of the MSP Shared Facility services is provided here as well as the services provided by the Targeted Metabolomics and Proteomics Laboratory (TMPL) services, here.