• Application Note

Absolute Quantitation of RapiFluor-MS Labeled N-Glycans by Calibration of Fluorescence Response

Absolute Quantitation of RapiFluor-MS Labeled N-Glycans by Calibration of Fluorescence Response
  • Qi Wang
  • Matthew A. Lauber
  • Waters Corporation
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Application Note

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This technology brief demonstrates the absolute quantitation of RapiFluor-MS labeled N-glycans using the RapiFluor-MS Quantitative Standard. 

Benefits

Together, the GlycoWorks RapiFluor-MS N-Glycan Kit and RapiFluor-MS Quantitative Glycan Standard provide a powerful new approach to quantitatively analyze N-glycans for proficiency testing, and to facilitate glycan-centric biomarker, as well as DMPK, studies.

Introduction

Glycosylation plays an important role in the structure and function of proteins, and altered glycosylation is implicated in numerous diseases, such as cancer,1,2 autoimmune diseases,3 and neurodegenerative disorders.⁴ Furthermore, glycosylation is frequently determined to be a critical quality attribute for therapeutic glycoproteins.5,6 Just like characterization, quantitation of protein glycosylation is needed for various reasons, including the discovery of glycan biomarkers for diagnosis, prognosis, or risk prediction,⁷ and drug metabolism and pharmacokinetics (DMPK) studies.⁸ Quantitative measurements are also essential to assessing the proficiency of an analytical method, where they can be helpful in estimating yields, facilitating validation studies, and confirming the success of transferring a method to a different laboratory. Recently, a novel analytical approach was introduced for the preparation and analysis of N-glycans. 

The strategy of this approach is based on combining the rapid release and labeling of N-glycans via the GlycoWorks RapiFluor-MS N-Glycan Kit with high-resolution hydrophilic interaction chromatography (HILIC) and sensitive fluorescence (FLR)-mass spectrometric (MS) analyses.⁹ In this work, we expand upon the capabilities of this approach and show how the RapiFluor-MS Quantitative Glycan Standard can be used to calibrate fluorescence response and thereby enable the absolute quantitation of RapiFluor-MS labeled N-glycans.

Results and Discussion

The RapiFluor-MS Quantitative Glycan Standard (p/n: 186008791) was purposefully designed to help analysts calibrate fluorescence responses observed during HILIC-FLR-MS separations of RapiFluor-MS labeled glycans. It is a synthetic peptide uniquely constructed of hydrophilic residues. Most importantly, it is derivatized by one mole equivalent of the RapiFluor-MS label and rigorously tested to ensure applicability to quantitative measurements. With a HILIC glucose unit (GU) value of approximately 4, it can be used as either an internal or external calibrant for N-glycan quantitation without interference issues from sample matrix void peaks or RapiFluor-MS labeled N-glycans, as demonstrated in Figure 1A–D. Since fluorescence response is linearly proportional to the amount of RapiFluor-MS label, fluorescence peak areas can be used to quantify specific RapiFluor-MS labeled glycans. For example, a linear regression can be graphed to relate observed fluorescence peak areas to different injected amounts of the RapiFluor-MS Quantitative Glycan Standard. Analyses have shown that a 4-point calibration curve consisting of 0.4, 0.8, 2.4, and 4.0 pmol of the standard provides an R² value of 1.0000. A quick 2-point (0.4 and 4.0 pmol) calibration curve for RapiFluor-MS labeled species can therefore be used to perform reliable absolute quantitation (Figure 1E). 

Among the different reasons to perform a quantitative analysis, one is to check the proficiency of a GlycoWorks RapiFluor-MS N-Glycan Kit (p/n: 176003713) sample preparation. In such a case, a control standard, like the Intact mAb Mass Check Standard (p/n: 186006552), can be used to study proficiency and yields. Here, we have focused on the amounts of FA2 glycan obtained from the Intact mAb Mass Check Standard with and without HILIC solid phase extraction (SPE) cleanup, which we have calculated, from their measured fluorescence peak areas, to be 1.64 and 2.20 pmol (Figure 1F). Since 45% of the glycans released from the Intact mAb Mass Check Standard are known to correspond to the FA2 glycan, the yield of glycans released and labeled from the Intact mAb Mass Check Standard using the GlycoWorks RapiFluor-MS N-Glycan Kit with and without HILIC SPE cleanup can be calculated to be 71% and 96%, respectively (Figure 1F-G). As previously reported, this result confirms that a RapiFluor-MS N-glycan sample preparation is effectively lossless, save for the SPE step.⁹ The value of the SPE step should not, however, be discounted as its losses have been confirmed to be unbiased, and its use can improve the lifetime of the downstream HILIC column. 

HILIC-fluorescence chromatograms.
Figure 1. Quantitation of RapiFluor-MS labeled N-glycans. HILIC-fluorescence chromatograms of 0.4 pmol (A) and 4.0 pmol (D) of RapiFluor-MS Quantitative Glycan Standard and RapiFluor-MS labeled N-glycans obtained from Intact mAb Mass Check Standard with (B) and without  HILIC SPE cleanup (C) using the GlycoWorks RapiFluor-MS N-Glycan Kit. Glycans were profiled with an ACQUITY UPLC H-Class Bio System and a fluorescence detector using an ACQUITY UPLC Glycan BEH Amide Column, 130Å, 1.7 µm, 2.1 × 150 mm. A two-point external calibration curve was constructed with the RapiFluor-MS Quantitative Glycan Standard (E) to enable quantitation of the RapiFluor-MS labeled FA2 glycan obtained from the Intact mAb Mass Check Standard (F). (G) Calculation for the theoretical yield of the FA2 glycan obtained from Intact mAb Mass Check Standard. Analyses were performed in duplicate.

The above glycan yields were obtained with one vial of the RapiFluor-MS Quantitative Glycan Standard. With three different batches of RapiFluor-MS Quantitative Glycan Standard, the glycan yields of the same preparation of RapiFluor-MS labeled N-glycans ranged from 67% to 72% with HILIC SPE cleanup and 90% to 96% without SPE (Figure 2A). The displayed error bars correspond to the relative standard deviations of glycan yields obtained from three vials of RapiFluor-MS Quantitative Glycan Standard within the same batch. In sum, these findings demonstrate the noteworthy vial-to-vial and batch-to-batch reproducibility of the RapiFluor-MS Quantitative Glycan Standard. The average glycan yields obtained from the three batches of RapiFluor-MS Quantitative Glycan Standard were 70% with SPE and 94% without SPE (Figure 2B, preparation #1). In the same fashion, glycan quantitation was also performed on a second N-glycan preparation, and the yields were found to be 64% with SPE and 93% without SPE (Figure 2B, preparation #2). These data clearly demonstrate the high reproducibility of the GlycoWorks RapiFluor-MS N-Glycan Kit for glycan release and labeling. As they are, these results also provide evidence in support of the proficiency with which the GlycoWorks RapiFluor-MS N-Glycan Kit sample preparations were performed.

Yields of RapiFluor-MS labeled glycans obtained from Intact mAb Mass Check Standard.
Figure 2. Yields of RapiFluor-MS labeled glycans obtained from Intact mAb Mass Check Standard. RapiFluor-MS labeled glycans obtained from the Intact mAb Mass Check Standard with and without HILIC SPE cleanup using the GlycoWorks RapiFluor-MS N-Glycan Kit were quantified with three different batches of the RapiFluor-MS Quantitative Glycan Standard (A). Glycan quantitation was performed on two GlycoWorks RapiFluor-MS N-Glycan Kit preparations of the Intact mAb Mass Check Standard (B).

Conclusion

We have demonstrated the absolute quantitation of N-glycans through combining HILIC-FLR analyses with the use of the RapiFluor-MS Quantitative Glycan Standard. In this work, we defined an external calibration curve from the fluorescence responses obtained with two different injected amounts of the RapiFluor-MS Quantitative Glycan Standard. It was thereby possible to quantify the amounts of specific RapiFluor-MS labeled N-glycans. As applied to proficiency testing, use of the RapiFluor-MS Quantitative Glycan Standard made it possible to determine and compare the yields of two RapiFluor-MS N-glycan sample preparations. In turn, it was demonstrated that release, labeling, and SPE clean-up of glycans with the GlycoWorks RapiFluor-MS N-Glycan Kit is achieved with very high reproducibility. Together, the GlycoWorks RapiFluor-MS N-Glycan Kit and RapiFluor-MS Quantitative Glycan Standard provide a powerful new approach to quantitatively analyze N-glycans. Here, its utility for proficiency testing was highlighted, but it should also be noted that the RapiFluor-MS Quantitative Glycan Standard could be used to facilitate glycan-centric biomarker and DMPK studies. 

References

  1. Mechref, Y. et al. Defining putative glycan cancer biomarkers by MS. Bioanalysis 2012, 4 (20), 2457–2469.
  2. Ruhaak, L. R., Miyamoto, S. and Lebrilla, C. B. Developments in the identification of glycan biomarkers for the detection of cancer. Molecular & Cellular Proteomics 2013, 12 (4), 846–855.
  3. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A. and Dwek, R. A. Glycosylation and the immune system. Science 2001, 291 (5512), 2370–2376.
  4. Lefebvre, T. et al. Does O-GlcNAc play a role in neurodegenerative diseases? Expert Review of Proteomics 2005, 2 (2), 265–275.
  5. Dalziel, M., Crispin, M., Scanlan, C. N., Zitzmann, N. and Dwek, R. A. Emerging principles for the therapeutic exploitation of glycosylation. Science 2014, 343 (6166), 1235681.
  6. Beck, A., Wagner-Rousset, E., Ayoub, D., Van Dorsselaer, A. and Sanglier- Cianférani, S. Characterization of therapeutic antibodies and related products. Analytical Chemistry 2012, 85 (2), 715–736.
  7. Etxebarria, J. & Reichardt, N.-C. Methods for the absolute quantification of N-glycan biomarkers. Biochimica et Biophysica Acta (BBA)-General Subjects 2016, 1860 (8), 1676–1687.
  8. Cong, Y., Zhang, Z., Zhang, S., Hu, L. and Gu, J. Quantitative MS analysis of therapeutic mAbs and their glycosylation for pharmacokinetics study. PROTEOMICS-Clinical Applications 2015.
  9. Lauber, M. A. et al. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Analytical Chemistry 2015, 87 (10), 5401–5409.

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