Simple Approach for Improved LC–MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas

Wang, Shunhai; Xing, Tao; Liu, Anita P.; He, Zehong; Yan, Yuetian; Daly, Thomas J.; Li, Ning

doi:10.1021/acs.analchem.8b05846

Cited by 25 publications

(27 citation statements)

References 22 publications

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“…The stripper solutions were based on 40% ACN with various additives: 0.1% propionic acid (PA) and 0.1% formic acid (FA), 1% PA, and 1% PA and 2% sulfolane. Both PA and sulfolane have been described as capable of reducing TFA signal suppression and adduct formation (propionic acid [ 7 , 13 , 19 ] and sulfolane [ 24 ]). The corresponding protein total ion chromatograms and mass spectra are reported in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To address this, several post-column strategies to perform efficient LC-MS using TFA-containing mobile phases have been proposed. These approaches include dilution [12][13][14], electrophoretic mobility control [15], membrane-based dialysis [16,17], and exposure of the nebulized LC effluent to gas vapors (e.g., ACN, propionic acid) during ESI [18,19]. Most of these reports are limited to low-flow applications, use flow splitters, or require complicated setups, hindering their general use.…”

Section: Introductionmentioning

confidence: 99%

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Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

et al. 2021

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Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation. Graphical abstract

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

et al. 2021

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“…Further improvements of this methodology are possible, e.g. by integrating this approach with front-end separations (as has bene recently demonstrated in the analysis of PEGylated protein therapeutics 37 ), as well as by complementing limited charge reduction in the gas phase with solution-phase charge manipulation techniques. 38 Authors' contributions: I.K., J.P. and Y.Y.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

Yang¹,

Pawlowski²,

Carrick³

et al. 2021

Preprint

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Intact-mass measurements are becoming an increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS measurements provides an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein, but also the mass range populated by various proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation, with the glycan load of phenotype 2-1 being notably lighter. More detailed characterization of their glycosylation patterns is enabled by the recently introduced crosspath reactive chromatography (XP-RC) with on-line MS detection, a technique that combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains, and provides a wealth of information on glycosylation patterns of the heavy chains. The haptoglobin 1-1 glycans are mature fully sialylated biantennary structures that exhibit high degrees of fucosylation. In contrast, phenotype 2-1 contains a significant fraction of incomplete biantennary structures and exhibit significantly lower levels of sialylation and fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of haptoglobin analysis by limited charge reduction, suggesting that the latter can be employed in situations when a fast assessment of a protein heterogeneity is needed (e.g., comparability studies of biopharmaceutical products). <br>

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“…The use of additives is one of the most effective and simple methods of increasing metabolite coverage. Alkylamine additives are widely used in LC-MS analysis of high-molecular-weight compounds [16,17,18,19,20,21]. The protocol design for the detection of high-molecular-weight compounds allows for neglecting the suppression effect of alkylamines that have accumulated on MS devices.…”

Section: Discussionmentioning

confidence: 99%

“…Secondary and tertiary alkylamines are commonly used as additives in the analysis of different classes of compounds (nucleosides, proteins, phospholipids, nucleic acids, etc.) in ESI(−) [16,17,18,19,20,21]. Unfortunately, there is no information about the application of alkylamines in untargeted metabolomic analysis.…”

Section: Introductionmentioning

confidence: 99%

n-Butylamine for Improving the Efficiency of Untargeted Mass Spectrometry Analysis of Plasma Metabolite Composition

Maslov

Trifonova

Balashova

et al. 2019

IJMS

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A comparative study of the impact of n-butylamine and traditionally used additives (ammonium hydroxide and formic acid) on the efficiency of the electrospray ionization (ESI) process for the enhancement of metabolite coverage was performed by direct injection mass spectrometry (MS) analysis in negative mode. Evaluation of obtained MS data showed that n-butylamine is one of the most effective additives for the analysis of metabolite composition in ESI in negative ion mode (ESI(−)) The limitations of the use of n-butylamine and other alkylamines in the analysis of metabolic composition and a decontamination procedure that can reduce MS device contamination after their application are discussed. The proposed procedure allows the performance of high-sensitivity analysis of low-molecular-weight compounds on the same MS device in both polarities.

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Simple Approach for Improved LC–MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas

Cited by 25 publications

References 22 publications

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

n-Butylamine for Improving the Efficiency of Untargeted Mass Spectrometry Analysis of Plasma Metabolite Composition

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