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. 2022 May 19;12:8433. doi: 10.1038/s41598-022-12423-x

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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HIC-UV chromatograms and RPLC-mass spectrometry analysis distinguishing CAP256V2LS sulfation proteoforms. Traces on the left are CAP256V2LS analyzed by HIC-UV. (y-axis is the UV absorbance, normalized to the highest peak intensity in each chromatogram). On the right, deconvoluted Reversed phase coupled with intact mass analysis from each peak fraction corresponding to the left elution after deglycosylation. (y-axis is the mass intensity, normalized to the highest peak in each mass spectrum.) (a) Unfractionated CAP256V2LS showed five peaks on HIC-UV chromatograms, with the peak of 4-SO₃ and 3-SO₃ species detected in RPLC-MS. (b) 4-SO₃ eluted first at 13.7 min with mass confirmed at 150,526.5 Da. (c) 3-SO₃ eluted at 15.6 min with mass of 150,447.3 Da. (d) 2-SO₃ eluted at 17.2 min with mass observed at 150,366.9 Da. (e) 1-SO₃ eluted at 18.7 min with mass detected at 150,287.2 Da. (f) 0-SO₃ eluted last at 20.1 min with mass measured at 150,206.4 Da.