US-12624065-B2 - Use of neutral pH mobile phases in reversed phase chromatography of acidic peptides

US12624065B2US 12624065 B2US12624065 B2US 12624065B2US-12624065-B2

Abstract

The present disclosure discusses a method of separating and/or purifying acidic peptides by the use of a mobile phase having a pH greater than or about equal to the isoelectric point of one or more of the metal oxides in the flow path.

Inventors

Samantha Ippoliti
Ying Qing Yu

Assignees

WATERS TECHNOLOGIES CORPORATION

Dates

Publication Date: 20260512
Application Date: 20220706

Claims (12)

1 . A method of separating sialylated O-glycopeptides, the method comprising: injecting a sample comprising a plurality of O-sialylated glycopeptides into a chromatographic system, the chromatographic system comprising a flow path, wherein at least a portion of the flow path comprises one or more metal oxides, wherein the O-sialylated glycopeptides have three or more sialylation sites per peptide; and flowing the sample through the chromatographic system under reverse phase chromatography conditions to separate the plurality of O-sialylated glycopeptides using a mobile phase having a pH of about 6.5 to about 8.5.
2 . The method of claim 1 , wherein the mobile phase comprises an acetate salt dissolved in water.
3 . The method of claim 1 , wherein the mobile phase comprises a buffered aqueous solution.
4 . The method of claim 1 , wherein the mobile phase has a pH between about 7 and about 8.
5 . The method of claim 1 , further comprising passing a fluid stream exiting the chromatographic column to a detector, wherein the fluid stream comprises one or more of the acidic peptides.
6 . The method of claim 5 , further comprising adding an acidic solution to the fluid stream prior to passing the fluid stream to the detector.
7 . The method of claim 6 , wherein the acidic solution comprises formic acid.
8 . The method of claim 6 , wherein the acidic solution has an acid concentration of between about 1% and 20% by volume in water.
9 . The method of claim 6 , wherein the ratio of the flow rate of the acidic solution to the flow rate of the fluid stream is between about 1:100 and about 1:5.
10 . The method of claim 5 , wherein the detector is a mass spectrometer.
11 . The method of claim 1 , wherein at least a portion of the flow path is coated with a low-bind surface coating.
12 . The method according to claim 11 , wherein the low-bind surface coating comprises an alkylsilyl coating.

Description

This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 63/218,694 filed on Jul. 6, 2021 and entitled “Use of Neutral pH Mobile Phases in Reversed Phase Chromatography of Acidic Peptides,” the contents of which are incorporated herein in their entirety. FIELD OF THE TECHNOLOGY The present disclosure relates to the use of a neutral pH mobile phase for improved chromatography and sample analysis of acidic peptides. BACKGROUND Analytes that interact with metals have often proven to be very challenging to separate. The desire to have high pressure capable chromatographic systems with minimal dispersion has required that flow paths decrease in diameter and be able to withstand increasingly high pressures at increasingly fast flow rates. As a result, the material of choice for chromatographic flow paths is often metallic in nature. This is despite the fact that characteristics of certain analytes, for example, small molecule pharmaceutical agents, proteins, peptides, glycopeptides, phosphopeptides, oligonucleotides, pesticides, bisphosphonic acids, anionic metabolites, and zwitterions like amino acids and neurotransmitters, are known to have unfavorable interactions, so called chromatographic secondary interactions, with metallic surfaces. Significant loss of acidic peptide analytes, such as phosphopeptides and sialylated glycopeptides, is observed when using conventional reversed phase liquid chromatography (RPLC)—mass spectrometry (MS) methods. This is believed to be due to chelation interactions between the acidic peptides and the column/system hardware. Traditional RPLC-MS peptide mapping chromatographic methods typically use C18 column chemistry with mobile phases generally comprising water, acetonitrile, and formic acid. The formic acid is added to the mobile phase to boost the electrospray ionization (ESI+) of analytes, increasing the sensitivity of mass spectrometry detection. Unfortunately, under acidic pH mobile phase conditions, the acidic peptides interact with metal oxides in the liquid chromatography (LC) flow path via chelation reactions. The binding strength to metals in the LC flow path increases as the number of acidic residues on the peptide increases. In the case of sialylated O-glycopeptides, when the sialyation is greater than 2 per peptide, traditional RPLC-MS chromatographic methods cannot recover the analytes from the column. Glycopeptides and phosphopeptides, therefore, suffer significant or complete loss of signal in a typical RPLC-MS analysis. For the sialylated glycopeptides, the peptide mass spectrometry signals can be recovered after removing the sialic acids by enzymatic treatment. However, such sample treatment leads to missed information on the sialic acid location and quantity. An alternative to using metal flow paths is to use flow paths constructed from polymeric materials, such as polyether ether ketone (PEEK). PEEK tubing, like most polymeric materials, is formed by means of an extrusion process. This manufacturing process can lead to highly variable internal diameters. Accordingly, PEEK column hardware yields unfavorable differences in the retention times as can be observed from switching between one column and the next. Often, this variation can be a factor of three higher than a metal constructed column. Ongoing efforts to reduce chelation and secondary chromatographic interactions of acidic peptides with metal oxides in chromatographic flow paths, in an effort to facilitate chromatographic separation having higher resolutions, are therefore needed. SUMMARY Problems associated with the analysis of acidic peptides can be resolved by the use of a mobile phase having a pH greater than or about equal to the isoelectric point of one or more metal oxides in the flow path. The use of such a mobile phase is believed to reduce the amount of positively charged metal oxides in the flow path, which inhibits potential ion exchange reactions between the acidic peptides and the metal oxides. This, in turn, leads to a reduction in loss of acidic peptide samples. In one embodiment, a method of separating and/or purifying acidic peptides comprises injecting a sample comprising one or more acidic peptides into a chromatographic system. The chromatographic system comprises a flow path. At least a portion of the flow path comprises one or more metal oxides. The sample is flowed through the chromatographic system under reverse phase chromatography conditions using a mobile phase having a pH greater than or about equal to the isoelectric point of one or more of the metal oxides in the flow path. Use of a mobile phase having a pH greater than or about equal to the isoelectric point of one or more of the metal oxides in the flow path allows acidic peptides to be analyzed using RPLC. Exemplary acidic peptides that can be analyzed using this technique include, but are not limited to, sialylated glycopeptides and phosphopeptides. In an embodiment, the mobi