Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent

Abstract

Protein glycosylation is a common protein post-translational modification (PTM) in living organisms and has been shown to associate with multiple diseases, and thus may potentially be a biomarker of such diseases. Efficient protein/glycoprotein extraction is a crucial step in the preparation of N-glycans derived from glycoproteins prior to LC-MS analysis. Convenient, efficient and unbiased sample preparation protocols are needed. Herein, we evaluated the use of sodium deoxycholate (SDC) acidic labile detergent to release N-glycans of glycoproteins derived from biological samples such as cancer cell lines. Compared to the filter-aided sample preparation approach, the sodium deoxycholate (SDC) assisted approach was determined to be more efficient and unbiased. SDC removal was determined to be more efficient when using acidic precipitation rather than ethyl acetate phase transfer. Efficient extraction of proteins/glycoproteins from biological samples was achieved by combining SDC lysis buffer and beads beating cell disruption. This was suggested by a significant overall increase in the intensities of N-glycans released from cancer cell lines. Additionally, the use of SDC approach was also shown to be more reproducible than those methods that do not use SDC.

Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample

“…A significant amount of protein loss happens when proteins are recovered from the spin filter membranes. Several studies have been done utilizing SDC during gel or in‐solution digestion sample preparations [21–26]. Besides, the application of SDC to plasma proteomics sample preparations was done successfully [27,28].…”

A rapid method for relative quantification ofN-glycans from a therapeutic monoclonal antibody during trastuzumab biosimilar development

“…MWCO filters were found to selectively bind certain glycans, and a detergent wash step (sodium deoxycholate, SDC) was added to the method (MWCO-SDC-IPC), which has been shown to be an improvement in filter-aided sample preparation in protein and glycoprotein digestion. 39,40 In addition, we used the PA-IPC method, which reliably identifies, quantifies and recovers more glycans, to compare the glycan profiles of innovator Herceptin® antibody products (6 lots, HER1 to HER6; 4 European and 2 US) with internal developmental biosimilar drug substance (scale-up bioreactor runs).…”

Advances in mass spectrometry‐based glycomics

“…and Zhu et al. . In case of the former , cell lysate was transferred into the filter membrane, and sodium dodecyl sulfate (SDS) was used as a detergent.…”

“…N‐ glycan profiles of wild‐type and mutant Chinese hamster ovary cell lysates suggested that this method referred to as filter‐aided‐ N‐ glycan separation (FANGS) is not biased to any N‐ glycan . More recently, Mechref and coworkers compared PNGase F digestion efficiency with and without sodium deoxycholate (SDC) . SDC is a detergent that was first introduced in proteomics studies to enhance protein solubilization, allowing the easy removal of the detergent by acid precipitation before MS analysis.…”

“…SDC is a detergent that was first introduced in proteomics studies to enhance protein solubilization, allowing the easy removal of the detergent by acid precipitation before MS analysis. Adding SDC to PNGase F digestion protocol prompt, efficient solubilization of membrane glycoproteins and the efficient release of their N‐ glycans . Both methods improved detergent removal steps, which are critical for MS analysis.…”