Product Information

PNGase F PRIME™ Glycosidase

 

Specifically removes N-linked oligosaccharides from glyco proteins

PNGase F PRIME™ (PRIME™) is a mutant recombinant PNGase F cloned from Flavobacterium meningosepticum and expressed and purified from E. coli. The proprietary changes made to PNGase F have been shown to have unique characteristics when compared to other commercially-available sources of PNGase F. Data generated by independent labs shows that PRIME™ works on native glycoproteins and serum glycoproteins in minutes at room temperature. Glycan analysis of the digestion products shows that PRIME™ digestion led to a more complete glycan release and also allowed for the cleavage of glycans not normally released by the commercially-available enzymes when used at the same concentrations with the same digestion conditions. This advancement benefits applications that seek to understand Glycobiology in a natural milieu. Preliminary data indicates that PRIME™ has a higher specificity towards complex (tri and tetra-antennary) sialylated structures compared to the commercially sourced enzyme.

 

PLEASE NOTE: For general users who are not Glycobiologists, every glycoprotein substrate is different.  Therefore results can vary depending on the target protein.

 

MALDI-IMS Analysis of a Mouse Brain Slice

Analysis by mass spectrometry is where PRIME™ truly shines. The three starred oligosaccharide groups in the mass spec trace to the right are color coded and overlaid on a mouse brain section. The distribution shows a distinct localization pattern.

As indicated, PNGase F Prime™ displays faster kinetics and has greater activity against native proteins as compared to commercially available enzyme. These features therefore make PNGase F Prime™ the enzyme of choice for use in MALDI-Imaging as this methodology requires a fast acting enzyme (as sprayed enzyme has the potential to dry on the slide) and the ability of the enzyme to work on native proteins (as found on cell surface).

As further information, the work presented in the following Analytical Chemistry paper utilized PNGase F PRIME™ for all in situ tissue work as other commercially-available PNGase enzymes did not work on native tissue to allow glycan recognition.

 

Powers TW, Jones EE, Betesh LR, Romano PR, Gao P, Copland JA, Mehta AS, Drake RR. (2013). Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues. Analytical Chemistry. 85:(20):9799-806.

MALDI Imaging MS Workflow [Analytical Chemistry 9-2013]
MALDI Imaging MS Workflow for Spatial Profiling Analysis of N-Linked Glycan Expression in Tissues
Publication Analytical Chemisty - MALDI-[...]
Adobe Acrobat document [354.1 KB]

 

Results from HPLC Analysis of Avastin® and Fetuin deglycosylation with PNGase F PRIME™

Traditional PNGase F yields fewer and more blunted peaks as compared with PRIME™. In nearly every side-by-side study to date, PRIME™ produces favorable results in this type of application. For example, native Avastin® (IgG1 based therapeutic for cancers) treated with PRIME™ releases 11 different oligosaccharides structures.

In another example, when compared with the commercially-available PNGase F enzyme, PRIME™ produced 33% more peaks with sharper peaks and higher signal to noise ratio when cleaving the native form of the blood borne glycoprotein fetuin.

The same treatments show marked differences even by HPLC analysis. PRIME™ (in blue) shows more pronounced peaks than the same competing PNGase F (in black). Also note the additional peaks in both the neutral and sialylated regions, particularly the trisialo region.

 

N-glycan imaging of a prostate cancer FFPE tissue.

A 5 micron FFPE slice of a human prostate cancer (Gleason 7) was antigen retrieved and incubated with PRIME™ as described in Powers et al. Images were acquired using a 7T MALDI-FTICR Solarix mass spectrometer and FlexImaging 4.1 software. Shown in the upper right panel is a H&E stained image of the tissue (at 10X magnification), with the area of the highest density of tumor shown in the circle, marked with a T. The bottom left panel shows a typical tumor N-glycan pattern for a single glycan, an Man6 at m/z=1419. In the top right panel and bottom right panel, a two-glycan overlay is shown for the tumor glycan at m/z = 1419 (in green) and a mono-sialylated biantennary glycan at m/z = 1976 (top right panel, orange) and a mono-sialylated and core fucosylated glycan at m/z = 2122 (bottom right panel, blue).

 

The following citations provide much greater detail of this research that utilized PRIME™:

 

Powers TW, Neely BA, Shao Y, Tang H, Troyer DA, Mehta AS, Haab BB, Drake RR. (2014) MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays. PLoS One. 9(9):e106255

 

Powers, T.W. Holst, S., Wuhrer, M.,Mehta, A.S., Drake, R.R. (2015) Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry. Biomolecules, 5, 2554-2572.

 

 

PNGase F PRIME Glycosidase

 

Specifically removes oligosaccharides from proteins

 

PNGase F PRIME™ is a mutant recombinant PNGase F cloned from Flavobacterium meningosepticum and expressed and purified from E. coli. The proprietary changes made to PNGase F have been shown to have unique characteristics when compared to other commercially-available sources of PNGase F. Data generated by independent labs shows that PRIME™ works on native glycoproteins and serum glycoproteins in minutes at room temperature. Glycan analysis of the digestion products shows that PNGase F PRIME™ digestion led to more complete glycan release and also allowed for the cleavage of glycans not released by the commercially-available enzymes when used at the same concentrations with the same digestion conditions. This advancement benefits applications that seek to understand glycobiology in a natural milieu. Preliminary data indicates that PNGase F PRIME™ has a higher specificity towards complex (tri and tetra-antennary) sialylated structures compared to the commercially sourced enzyme. Additionally, the work presented in this Analytical Chemistry paper utilized PNGase F PRIME™ for all in situ tissue work as the commercially-available PNGase enzymes did not easily work on native tissue to allow glycan recognition.

 

MALDI-IMS Analysis of a Mouse Brain Slice

 

PNGase Treated Sample on Mass Spec

 

 

 

 

 

 

 

 

 

 

 

 

The three starred oligosaccharide groups in the mass spec trace above are color coded and overlayed on a mouse brain section. The distribution shows a distinct localization pattern.

 

Analysis by mass spectrometry is when PRIME™ makes a difference. Traditional PNGase provides fewer and blunted peaks as compared with PRIME™. In nearly every side-by-side study conducted to date, PNGase F PRIME™ produces favorable results. For example, native Avastin® (IgG1 based therapeutic for cancers) treated with PRIME™ releases 11 different oligosaccharides structures (only a subset of data shown below).

 

Avastin treated with PNGase F PRIME

 

In another example, when compared with a very popular PNGase F, PRIME™ produced 33% more peaks, sharper peaks and with much lower background when cleaving the native form of the blood borne glycoprotein fetuin.

 

 

The same treatments show marked differences even by HPLC analysis. PNGase F PRIME™ (in blue) shows more pronounced peaks than the same competing PNGase F (in black). Also note the additional peaks in both the neutral and sialylated regions, particularly the trisialo region.

 

 

This type of richer data presents the opportunity to develop a deeper, more complete, understanding of glycobiology.

 

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