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. 2025 Jul 18;5(4):559–571. doi: 10.1021/acsmeasuresciau.5c00047

Absolute Quantitation of Phosphopeptides and Glycopeptides Using Coulometric Mass Spectrometry

Md Tanim-Al Hassan †, Yongling Ai †, Bhavya Deshaboina †, Timothy Yaroshuk †, Arjun Sharma †, Quentin Young †, Howard D Dewald ‡, Hao Chen †,*
PMCID: PMC12371591  PMID: 40861915

Abstract

Phosphorylation and glycosylation are two important protein post-transitional modifications (PTMs). However, quantification of these PTMs is challenging due to the lack of protein or peptide standards. In this study, we introduced a novel approach using coulometric mass spectrometry (CMS) for absolute quantitation of phosphopeptides and glycopeptides without using standards. First, phosphorylated tyrosine peptides such as TSTEPQpYQPGENL and RRLIEDAEpYAARG can be converted into electrochemically active tyrosine peptides via enzymatic phosphate removal using alkaline phosphatase prior to CMS quantitation. Accurate quantitation was obtained with small quantitation errors (0.3–6.6%). Alternatively, for electrochemically inactive phosphopeptides and glycopeptides, derivatization of their N-termini with an NHS ester reagent, 2,5-dioxo-1-pyrrolidinyl 3,4-dihydroxybenzene propanoate (DPDP), was conducted to introduce one electroactive catechol tag, allowing the DPDP-derivatized peptides to be quantified by CMS. This strategy was first validated using peptides RGD, GGYR, phosphopeptide RRApSVA, and glycopeptide NYIVGQPSS­(β-GlcNAc)­TGNL–OH, and successful quantification was achieved with quantification errors less than 6%. Taking one step further, we applied this approach to quantify glycopeptides generated from tryptic digestion of the NIST monoclonal antibody (mAb). Through hydrophilic interaction liquid chromatography column separation, five N297 glycopeptides were successfully derivatized, separated, and quantified by CMS without the use of standards. Due to the biological significance of PTMs, this study for quantifying peptides carrying PTMs would have a high potential for quantitative proteomics and biological research.

Keywords: phosphopeptide, glycopeptide, post-translational modification, absolute quantitation, electrochemistry, mass spectrometry

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Introduction

Phosphorylation and glycosylation are among the most common and extensively studied post-translational modifications (PTMs). − About 30% of the human proteome is phosphorylated, and phosphoproteins may exist as multiple phosphorylated forms. There are two main kinds of phosphorylation: O-phosphorylation, which occurs on serine, threonine, and tyrosine residues, and N-phosphorylation, which takes places on histidine, arginine, and lysine. Glycosylation plays various roles in the structures and functions of proteins, and alterations in glycosylation could lead to numerous diseases including cancer and immune system deficiencies. − N-glycosylation occurs mainly at asparagine (Asn or N) residues, where the consensus tripeptide sequence NXS/T is located (where X is any amino acid except proline). At least one N-glycosylation motif is present in two-thirds of all human proteins, and about 50% of all human proteins are N-glycosylated. Other types of glycosylation include O-glycosylation, which occurs in the serine and threonine residues, and C-glycosylation, which bonds on the indole rings of the tryptophan residues. ,

In proteomics research, quantitative information on peptides and proteins is critical for helping us understand biological processes. Quantification in mass spectrometry (MS)-based proteomics research relies on two fundamental strategies: relative and absolute quantitation. − Relative quantitation methods involve comparing the levels of peptides or proteins among different samples. A traditional label-free approach involves analyzing samples and comparing their mass spectra to determine peptide abundances relative to another sample. , Alternatively, isotopic labeling is commonly used in relative quantitation, where peptides from two samples are labeled with heavy and light isotopes, respectively. , The intensities of these labeled peptides are then compared to discern changes in abundance between the samples. ,− Elegant approaches like tandem mass tag, isotope-coded affinity tags, stable isotope labeling by amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantitation, metal element chelated tags, isotope-coded protein labeling, and N,N-dimethyl leucine (DiLeu) isobaric tag methods fall under this category. − Likewise, absolute quantitation of proteins involves spiking a known quantity of isotope-labeled target peptide or protein into an experimental sample, followed by LC–MS or LC–MS/MS analysis. ,,,− The three most well-known methods for absolute quantitation are absolute quantification (AQUA), quantification conCATamer (QconCAT), and protein standard absolute quantification. While these elegant techniques offer the advantage of determining the absolute amounts of target peptides across different samples, the necessity of using standard or isotope-labeled peptides poses challenges. These standard peptides may not always be readily available, and their synthesis can be expensive and time-consuming. ,, Thus, the quest for a standard-free absolute quantitation method remains an ideal goal in proteomics research. This is particularly true for quantifying protein PTMs such as phosphorylation and glycosylation, in which the standard phosphopeptides or glycopeptides may be even more difficult to obtain.

Recently, we developed coulometric mass spectrometry (CMS) for absolute quantitation of electroactive analytes, using a liquid chromatography (LC)/electrochemistry/MS (LC/EC/MS) apparatus (illustrated in Figure S1). − ,− The method is based on electrochemical oxidation/reduction of analytes, followed by MS to measure the oxidation/reduction yield. Electrochemical reactions result in an electric current response, which can be integrated over time to calculate the electric charge Q involved in the redox reaction. According to Faraday’s law, Q is proportional to the quantity of the oxidized/reduced analyte: Q = nzF, where n is the mole of the oxidized/reduced analyte, z is the number of electrons transferred per molecule during the redox reaction, and F is the Faraday’s constant (9.65 × 104 C/mol). Therefore, the moles of the oxidized/reduced analyte can be calculated as n = Q/zF. Meanwhile, upon oxidation or reduction, the target analyte shows a reduced intensity in the acquired MS spectra, and the relative analyte ion intensity change, Δi, reflects the redox conversion yield. Thus, the moles of the oxidized/reduced analyte, in combination with the conversion yield, can be used to calculate the total amount of the analyte. In other words

Totalamountoftheanalyte=(amountoftheoxidized/reducedanalyte)/(theconversionyield)
=(amountoftheoxidized/reducedanalyte)/(theconversionyield)
=Q/(zFΔi) 1

With this CMS method, we conducted absolute quantitation of peptides and proteins based on electrochemical oxidation of oxidizable cysteine, tyrosine, and tryptophan residues. − ,− For peptides without oxidizable residues, they could be derivatized to carry an electrochemical tag such as methylene blue, allowing them to be quantifiable by CMS after derivatization. In this study, we extended the CMS quantitation approach for peptides with PTMs such as phosphopeptides and glycopeptides. For phosphorylated tyrosine peptides, their phosphates were removed enzymatically to generate electroactive tyrosine peptides (Scheme a), before CMS quantitation. Alternatively, for electrochemically inactive phosphopeptides or glycopeptides, peptide derivatization with DPDP (Scheme b) was performed to introduce one electroactive catechol tag, prior to CMS quantitation. Upon electrochemical oxidation at a low oxidation potential (0.7 V vs Ag/AgCl), the catechol tag tends to lose two protons and two electrons to form a quinone product (z = 2, Scheme b), facilitating CMS quantitation. Using these strategies, several phosphopeptides, glycopeptides, as well as glycopeptides from NIST mAb digest were successfully quantified, without using standards.

1. Our Approaches for Absolute Quantitation of a PTM-Modified Peptide by CMS (a) after Removing Phosphate Using Phosphatase and (b) after Derivatization of Its N-Terminal with DPDP.

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Experimental Section

Chemicals

N-terminal-protected amino acid BOC-Lys-OH, Gly-Gly-Tyr-Arg (GGYR, HPLC grade), Arg-Gly-Asp (RGD, HPLC grade), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were purchased from Sigma-Aldrich (St Louis, MO). Phosphorylated RRApSVA (HPLC grade) was obtained from EMD Millipore (Temecula, CA). RRLIEDAEpYAARG, TSTEPQpYQPGENL, alkaline phosphatase (1 KU) from bovine intestinal mucosa, and NIST mAb antibody were bought from Sigma-Aldrich (St. Louis, MO). NYIVGQPSS­(β-GlcNAc)­TGNL–OH was purchased from Sussex Research (ON, Canada). 2,5-Dioxo-1-pyrrolidinyl 3,4-dihydroxybenzene propanoate (DPDP) was bought from Aquila Pharmatech LLC (Waterville, OH). Trypsin (sequencing grade) was purchased from Promega (Madison, WI). Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), and sodium hydroxide were procured from Fisher Chemical (Fair Lawn, NJ). Formic acid (FA) and acetonitrile were obtained from Fisher Chemical (Fair Lawn, NJ), and deionized water used for sample preparation was obtained by using a Millipore purification system (Burlington, MA).

Instrument

Our CMS system (apparatus shown in Figure S1) consisted of LC, electrochemistry (EC), and MS. Basically, an ultraperformance LC (UPLC, Waters, Milford, MA) was connected with an electrochemical thin-layer flow cell equipped with either a glassy carbon electrode (GCD, 6 mm ID) or a boron-doped diamond electrode (BDD, 8 mm ID) as the working electrode (WE). The flow cell was further coupled to a high-resolution Orbitrap Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA). A Ag/AgCl (3 M NaCl) electrode (reference electrode (RE), catalog no. M W-2021, BASi, West Lafayette, IN) was employed as a RE, while the stainless-steel body of the cell functioned as the counter electrode (CE).

Separation of peptides was achieved using either an Agilent poroshell reversed-phase column (HPH–C18, 2.1 mm × 100 mm, 1.9 μm, Agilent, Santa Clara, CA), a reversed-phase column (BEH C18, 2.1 mm × 100 mm, 1.7 μm), or a hydrophilic interaction liquid chromatography (HILIC) column (HALO Penta HILIC column, 2.1 mm × 250 mm; 2.7 μm, used for the separation of NIST mAb glycopeptides). The mobile phase flow rate was set at 0.2–0.3 mL/min. Mobile phase A used was water with 0.1% FA, and mobile phase B was acetonitrile with 0.1% FA.

OriginPro 2018b was used to import and integrate the recorded electric current peak to calculate the total electric charge of Q. The peptide flowing out of the electrochemical cell was online analyzed using the Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI) source with the following parameters: sheath gas flow rate, 35; auxiliary gas flow rate, 10; spray voltage, 3.0 kV; sweep gas flow rate, 0; capillary temperature, 300 °C; auxiliary gas heater temperature, 100 °C; and S-lens RF level, 50. Mass spectra were acquired by a Thermo Xcalibur (3.0.63). Before each use, the glassy carbon WE underwent thorough cleaning by polishing with an alumina slurry. In situ cleaning involved applying alternative positive and negative potentials of + 2.0 V and – 2.0 V was used for the BDD electrode.

Cyclic Voltammetry Experiment

We first derivatized the GGYR peptide using DPDP and isolated the derivatized peptide, denoted as *GGYR (* represents the derivatized peptide), via fractionation by Shimadzu LC (LC 40B XR). Using a homemade undivided cell, we utilized 20 mL of 200 μM intact GGYR or 30 μM of *GGYR for the cyclic voltammetry (CV) experiments conducted in H2O:ACN:FA (70:30:1). A 3 mm GCE electrode with an area of 0.07 cm2 served as the WE, while a Ag/AgCl RE was positioned in close proximity to the WE to regulate the potential. Additionally, a carbon cloth of 5 cm2 size was positioned opposite to the WE, serving as the CE. Monitoring and recording of the oxidation current were facilitated by an Epsilon EClipse potentiostat (BASi, West Lafayette, IN, USA).

Alkaline Phosphatase Treatment

Phosphatase was dissolved in 1 mL of 10 mM tris–HCL (pH 8.0) containing 1 mM magnesium chloride and then stored in the fridge (4 °C). 50 μL of RRLIEDAEpYAARG (1 mM) or 100 μL of TSTEPQpYQPGENL (0.5 mM) was mixed with 20 μL of alkaline phosphatase (0.86 KU/mL) and incubated at 37 °C for 1 h. FA was added (final FA concentration: 1% by volume) after the dephosphorylation reaction. The sample was cooled to room temperature and diluted with 0.1% FA to a final concentration of 1.0 μM for CMS analysis.

Protein Digestion

A 40 μL portion of 10 μg/μL (400 μg) of NIST mAb (ref: 8671) was denatured using 80 μL of 8 M urea at room temperature and then vortexed for 15 min. Five μL of 1 M DTT (freshly prepared) was added to the sample and then shaken for 1 h at 37 °C in a water bath. A 20 μL portion of 500 mM IAA was then added to the sample at room temperature in the dark for 30 min. The sample mixture was then desalted and buffer-exchanged with 50 mM ABC buffer by an Amicon 10 K molecular weight cutoff filter (MWCOF) and then digested overnight with 20 μg of trypsin (protein/trypsin ratio: 20:1).

Peptide Derivatization with DPDP

For the proof-of-concept experiment, we first derivatized the N-terminal-protected amino acid BOC-Lys-OH with DPDP-NHS ester in a 1:10 molar ratio using H2O:ACN (50:50) as a solvent with pH 8.3 adjusted by NaOH. The sample was derivatized for overnight reaction using mild vortexing in dark. The same derivatization condition was applied for peptides RGD, RRApSVA, and NYIVGQPSS­(β-GlcNAc) TGNL–OH. To maintain a good buffering system for controlling pH and reduce reaction time, we also derivatized the GGYR peptide using 100 mM HEPES buffer (pH 8.3) and using a 1:10 molar ratio (peptide/DPDP) for the derivatization process.

For the derivatization of glycopeptides from mAb, the mAb digest sample was vacuum-dried and reconstituted in 20 mM HEPES buffer (pH 8.5) to prepare a 5.3 μM sample, which was then shaken with DPDP (159 mM, 3000-fold in excess) overnight in the dark for derivatization. The sample was dried out and reconstituted in ACN/H2O (65:35 v/v) to prepare a 5.3 μM derivatized sample. An underivatized sample was also prepared to assist in the measurement of the glycopeptide derivatization yields.

LC Separation for CMS Quantitation

For CMS quantitation of phosphopeptides RRLIEDAEpYAARG and TSTEPQpYQPGENL, after removal of phosphate, 10 μL of 1.0 μM peptide solution was injected by LC (10 pmol injection amount). The mobile phase flow rate was 200 μL/min. In a 10 min gradient elution, mobile phase B increased from 2% to 70% in 3 min and reached 90% in 1 min. Then, mobile phase B was kept at 90% for 3 min before returning to 2%.

For CMS quantitation of derivatized peptides, the LC elution program was similar, and the mobile phase flow rate was set 300 μL/min, and 5 μL of 1 μM derivatized peptides (5 pmol injection amount) was injected for analysis For the derivatized BOC-Lys-OH, the LC gradient elution program started with 90% A for 1 min, 90% A to 40% A in 8 min, linear gradient at 5% A for 2 min, and then 90% A for 2 min. For the derivatized RGD and phosphopeptide RRApSVA, the LC gradient elution program started with 90% A for 1 min, 90% A to 80% A in 3 min, linear gradient at 5% A for 1 min, and then 90% A for 1 min. For the derivatized NYIVGQPSS­(β-GlcNAc) TGNL–OH LC, the gradient elution program started with 95% A for 1 min, 95% A to 70% A in 10 min, linear gradient at 5% A for 3 min, and then 95% A for 2 min. For the derivatized GGYR, the mobile phase started with 95% A, 95% A to 91% A in 10 min, linear gradient at 5% A for 2 min, and then 95% A for 2 min. To avoid interference of the HEPES buffer in the GGYR sample, the first 3 min of eluate was directed to waste.

For CMS quantitation of derivatized mAb glycopeptides, the mobile phase flow rate was set at the 200 μL/min A gradient elution program starting with 25% A for 5 min (the first 5 min of eluate was directed to waste), 25% A to 35% A in 30 min, linear gradient at 60% A for 5 min, and then 25% A for 5 min. A 10 μL portion of the derivatized mAb digest (5.3 μM, injection amount 53 pmol) was injected for CMS quantitation.

Results and Discussion

The integration of MS with electrochemistry (EC), namely, EC/MS, has drawn considerable attention and found many applications, including drug metabolism study, protein structural analysis, and electrochemical reaction mechanism elucidation. − The method can also be used to capture elusive reaction intermediates − and enable electrosynthetic reaction screening. − As a new application of the EC/MS technique, the unprecedented advantage of CMS is the capability of absolute quantitation without using any standards or establishing a calibration curve. In our previous research, we successfully demonstrated the capability of CMS to quantify peptides containing electroactive residues like cysteine, tyrosine, and tryptophan. − ,− In this study, a similar CMS approach was adopted to quantify peptides containing phosphorylated tyrosine residues after enzymatic removal of phosphate to generate electrochemically active tyrosine peptides. As the removal of phosphate by phosphatase is quantitative, the measured quantity of the resulting tyrosine peptide can reflect the amount of the phosphopeptide precursor. On the other hand, in a more general approach, phosphopeptides or glycopeptides can be selectively derivatized by DPDP to carry an electrochemical tag of catechol (Scheme b), prior to CMS quantitation. In comparison to our previously used methylene blue tag, the catechol tag is more polar and water-soluble, leading to better peptide separation by LC and therefore benefits CMS quantitation.

CMS Quantitation of Phosphopeptides after Phosphate Removal

To explore the feasibility of CMS quantitation of phosphorylated tyrosine peptides, one standard peptide TSTEPQpYQPGENL was first tested. As shown in Figure a,b, after incubation with alkaline phosphatase, the TSTEPQpYQPGENL was quantitatively − converted into TSTEPQYQPGENL (the original peak of TSTEPQpYQPGENL at 3.2 min disappeared (Figure a); instead, the product TSTEPQYQPGENL peak appeared at 3.5 min in the TIC diagram (Figure b). When the intact TSTEPQpYQPGENL was injected, no electric current peak was observed with an applied voltage of + 1.05 V for oxidation (Figure g), indicating that phosphorylated tyrosine is not electrochemically active. In contrast, after the injection of the dephosphorylated peptide TSTEPQYQPGENL sample, a sharp electric current peak was observed under the same oxidation conditions (Figure h), which is in line with our previous observation of electrochemical oxidation of tyrosine-containing peptides. , Indeed, this was further confirmed by the corresponding MS spectra recorded upon electrochemical oxidation (Figure c,d). Compared to the MS spectrum (Figure c) without electrolysis, the +2 ion of TSTEPQYQPGENL was observed at m/z 731.84, whereas a new peak at m/z 730.84 (Figure d), corresponding to the +2 ion of the oxidized peptide product and resulting from 2 electron and 2 proton losses upon tyrosine oxidation, ,, was detected, when +1.05 V was applied to the cell for oxidation. The integrated extracted ion chromatogram (EIC) peak area of m/z 731.84 shown in Figure f was reduced by 18.4% compared to the same peak in Figure e, suggesting that the oxidation yield for TSTEPQYQPGENL was 18.4% (see the detailed data in Table S1). The amount of the oxidized TSTEPQYQPGENL was calculated to be 10.64 pmol based on the integration of the current peak area shown in Figure h and the oxidation yield using eq . Therefore, the measured amount of TSTEPQYQPGENL was 10.64 pmol (Table S1). A triplicate measurement gave the average amount of TSTEPQYQPGENL to be 10.66 pmol, which turned out to be close to the theoretical amount of 10.0 pmol, with a measurement error of 6.6% (Table S1).

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TICs of TSTEPQpYQPGENL (a) before and (b) after adding phosphatase. MS spectra of TSTEPQYQPGENL (c) when the cell was off and (d) when the cell was turned on (applied potential: +1.05 V). The oxidation product of TSTEPQYQPGENL was detected at m/z 730.84. EICs of TSTEPQYQPGENL at m/z 731.84 were acquired (e) when the cell was off and (f) when the cell was turned on (applied potential: +1.05 V). Electric current diagrams were collected from (g) the oxidation of TSTEPQpYQPGENL and (h) the oxidation of TSTEPQYQPGENL.

RRLIEDAEpYAARG, another phosphopeptide that contains phosphotyrosine, was also analyzed in the same way by CMS. RRLIEDAEpYAARG was also quantitatively converted into RRLIEDAEYAARG by phosphatase (Figure S2a,b). No electric current was detected for RRLIEDAEpYAARG (Figure S2g), while a sharp current was observed for the dephosphorylated peptide RRLIEDAEYAARG sample (Figure S2h). The oxidation product was seen at m/z 506.27 after electrolysis (Figure S2d). The oxidation yield was measured by the comparison of the EIC peak area of m/z 506.94 before and after oxidation (Figure S2e,f). In a triplicate measurement, the averaged quantity of this peptide measured by CMS was 10.03 pmol, which is very close to the initial amount of the expected value of 10.00 pmol with a measurement error 0.3% (detailed data is shown in Table S2). The results shown above confirm that CMS can be used for absolute quantitation of phosphorylated tyrosine peptides, in combination with enzymatic phosphate removal.

CMS Quantitation after Derivatization

Quantitation of Standard Amino Acid and Peptides

In this study, we also developed an alternative CMS approach for quantifying phosphopeptides and glycopeptides, in combination with the DPDP derivatization strategy. First, we tested some model peptides to validate the quantitation method and then applied it to quantify phosphopeptides and glycopeptides.

We initially chose the N-terminal-protected amino acid BOC-Lys-OH as a model compound for our investigation. We observed a 94% reaction efficiency for this amino acid, as evidenced by a decrease in the EIC peak area of the intact peptide ion at m/z 247.1 from 5.80 × 106 before DPDP derivatization to 3.44 × 105 after DPDP derivatization (shown in Figure S3). For CMS quantitation, we injected 5 μL of 1 μM BOC-Lys-OH after its derivatization (injection amount: 5.0 pmol). Additionally, a minor potential of 0.7 V (vs Ag/AgCl) was applied to the WE of the flow cell to facilitate the sample oxidation. Prior to oxidation, the +1 ion of DPDP-derivatized BOC-Lys-OH was detected at m/z 411.2 (Figure S4a). Another peak at m/z 409.2 was also seen, probably due to in-source oxidation during sample ionization (Figure S4a). Upon electrochemical oxidation, the intensity of the +1 ion of the DPDP-derivatized BOC-Lys-OH decreased (Figure S4,b). Conversely, the intensity peak at m/z 409.2 increased (Figure S4,b) due to electrochemical oxidation. The EIC peak area of the DPDP-derivatized BOC-Lys-OH at m/z 411.2 decreased by 21% following oxidation (Figure S4,d) compared to its preoxidation EIC peak (Figure S4,c), indicating an oxidation yield of 21% (Table S3). The total charge involved in peptide oxidation was determined to be 1.67 × 10–7C through integration of the peptide oxidation current (Figure S4,f). According to eq and z = 2 for the oxidation of the catechol moiety, it was determined that the amount of BOC-Lys-OH by CMS was 4.4 pmol, after considering 94% derivatization yield. In triplicate measurements, the average value of the three runs was calculated to be 4.7 pmol (Table S3). The theoretical amount of BOC-Lys-OH used for CMS quantification was 5.0 pmol. The quantitation error of BOC-Lys-OH was −5.4% (Table S3), suggesting the quantitation feasibility of this alternative CMS approach.

Peptide RGD was then chosen as an additional test sample for CMS quantification method validation. A derivatization yield of 94% for this peptide using DPDP was measured using LC/MS in a similar way as described above for sample BOC-Lys-OH. Figure S5,a displays the +1 ion of the derivatized RGD (m/z 511.2) and its oxidized product (m/z 509.2, probably due to in-source oxidation). The EIC peak area of m/z 511.2 decreased by 10% following oxidation, indicating an oxidation yield of 10% (Figure S5c,d). The total charge Q during oxidation was recorded at 7.16 × 10–8C. A theoretical 5.0 pmol of the peptide was injected, with an actual measured amount of 4.7 pmol (calculated based on eq with further consideration of the 94% derivatization yield, see details in Table S4), reflecting a slight measurement error of −6%.

Another peptide GGRY was also selected and derivatized by DPDP for testing, for the reason to show that the catechol tag can be selectively oxidized without oxidizing its tyrosine residue when the applied potential is low. In order to gain deeper insights into the oxidation properties of the DPDP-derivatized peptide, we conducted CV of intact GGYR and the derivatized GGRY, *GGYR. *GGRY was obtained by the reaction of GGYR with DPDP followed by LC purification. A CV of 20 mL of 200 μM intact GGYR peptide (dissolved in H2O:ACN:FA, 70:30:1 by volume) was conducted. Upon initiating the CV experiment with a positive scan from 0 to 1400 mV (vs Ag/AgCl), a discernible peak (I) at a potential of 1050 mV corresponding to the oxidization of the tyrosine residue of GGYR was observed as shown in Figure a (the observed onset oxidation potential for tyrosine is 900 mV). The oxidation of tyrosine at 1050 mV is consistent with our previous observation. In contrast, CV of 20 mL of 30 μM *GGYR (dissolved in H2O:ACN:FA, 70:30:1 by volume) was performed, two peaks (I and II) were noticed, corresponding to the oxidation of the tyrosine moiety of *GGYR at 1050 mV (peak I, Figure b), and the oxidation of the catechol tag of *GGYR at 700 mV (the observed onset oxidation potential for the catechol tag is 620 mV, peak II, Figure b). A background CV in H2O:ACN:FA (70:30:1 by volume) marked by dashed lines in Figure a,b was also collected to separate faradaic current from its nonfaradic counterpart. From the CV plots shown in Figure a,b, it was clear that the selective oxidation of the tag of the derivatized peptide without oxidizing the tyrosine residue can be achieved at a potential range between 620 and 900 mV. Therefore, in our experiment, 700 or 800 mV was selected as the oxidation potential for our CMS experiments. It can be seen that our newly employed derivatizing agent DPDP offers a distinct advantage by undergoing oxidation at a lower potential of 0.7 V vs Ag/AgCl during CMS quantitation. This characteristic shows that it is less susceptible to the phenomenon of surface fouling on the electrode. Throughout our experimental endeavors, we observed a reduction in the frequency of electrode cleaning procedures.

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Cyclic voltammetric behaviors of (a) GGYR and (b) derivatized GGYR. Mass spectra of derivatized GGYR (c) before electrochemical oxidation and (d) after electrochemical oxidation at 0.7 V; EIC peaks of derivatized GGYR at m/z 616.2 (e) before oxidation and (f) after oxidation; diagram showing the electrochemical oxidation currents of (g) the blank solvent and (h) the derivatized GGYR sample.

In order to improve the efficiency of peptide derivatization using the DPDP reagent, 100 mM HEPES buffer (pH 8.3) was used as the buffer for GGYR derivatization, and a 100% derivatization yield was achieved (no intact GGYR was seen after derivatization for 4 h), thus avoiding the necessity of measuring the derivatization yield. This process also significantly shortened the reaction time compared to that of the traditional overnight protocol. Subsequently, *GGRY was subjected to CMS measurement. Before introducing the sample to the electrochemical cell, the first 3 min were diverted to waste to eliminate the HEPES buffer. When the cell was turned off, an ion of m/z 616.2 was observed (Figure c), corresponding to the protonated *GGRY. Another ion at m/z 614.2 was also seen (Figure c), probably due to in-source oxidation. The integrated EIC peak area for m/z 616.2 decreased by 7.2% following oxidation (Figure e,f), indicating a peptide oxidation yield of 7.2%. The total oxidation charge (Q) was calculated as 6.92 × 10–8 C (Figure g,h). We injected an expected 5.0 pmol of peptide with an actual measured amount of 4.7 pmol (triplicate measurements, Table S5), yielding a small measurement error of −5.4%.

To verify that the oxidation did occur to the catechol tag located in the N-terminus of the *GGRY peptide, both the oxidized peptide product ion (m/z 614.2) and the intact peptide ion (m/z 616.2) were subject to MS/MS analysis. For this analysis, the *GGYR sample was introduced into the electrochemical cell for oxidation at 0.7 V using a syringe pump. The oxidation product was collected for analysis by nanoelectrospray ionization, resulting in the detection of the oxidized G’GYR ion (’ indicates the oxidized catechol tag) at m/z 614.2. As depicted in Figure S6, upon collision induced dissociation, m/z 614.2 produced a fragment ion b2-2H (m/z 277.1), whereas the intact *GGYR ion (m/z 616.2) generated b2 (m/z 279.1), which is in line with the occurrence of oxidation to the N-terminal catechol tag of *GGYR indeed.

Our method’s quantitation sensitivity was also evaluated by investigating CMS quantitation by injecting 6 μL of 25 nM *GGYR sample (total amount: 150 fmol *GGYR). The integrated EIC peak area for m/z 616.2 decreased by 9.5% following oxidation (Figure S7c,d), indicating a peptide oxidation yield of 9.5%. The total oxidation charge (Q) was calculated as 2.54 × 10–9 C (Figure S7f). The actual measured amount of 146 fmol by CMS (triplicate measurements, Table S6) showed a small measurement error of −3.0% in comparison with the theoretical injection amount of 150 fmol.

Quantification of Phosphopeptides/Glycopeptides

After quantifying standard peptides, we applied the method to quantify phosphopeptides and glycopeptides, as a demonstration of its application. A phosphorylated peptide, RRApSVA, was selected as a test sample that had no oxidizable amino acid residue. The derivatization yield of this peptide was determined to be 68% by comparing the EIC peak area of the intact peptide (m/z 739.4) signal before and after the derivatization reaction (Table S7). Prior to oxidation, the +1 ion of derivatized RRAPSVA was detected at m/z 903.4 (Figure a). An in-source oxidation peak was seen at m/z 901.4 (Figure a), and its signal intensity increased after triggering the oxidation by applying 0.7 V (vs Ag/AgCl) to the WE of the cell (Figure b). In-source oxidation caused the measurement complexity due to an overlap of m/z 903.4 with the M + 2 isotope peak of m/z 901.4. To avoid this complexity, we selected the peak at m/z 904.4 to calculate the oxidation yield. Upon oxidation, the integrated EIC peak area for m/z 904.4 was reduced by 5.5% (Figure d) compared to before oxidation (Figure c), indicating an oxidation yield of 5.5%. The total charge Q involved in the oxidation in this case was determined to be 3.65 × 10–8C (Figure e,f). This Q value, along with the oxidation yield (i.e., eq ), was utilized to calculate the quantity of the derivatized peptide to be 3.43 pmol (Table S7). Considering the 68% derivatization yield, the total peptide amount was calculated to be 5.0 pmol. Triplicate measurements gave an average peptide amount of 5.2 pmol (Table S7). Once again, this value aligns well with the theoretical peptide amount of 5 pmol with a small quantitation error of 3.4% (Table S7).

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Mass spectra of the *RRApSVA (a) before electrochemical oxidation and (b) after electrochemical oxidation at 0.7 V; EIC peaks of the *RRApSVA ion at m/z 904.4 (c) before oxidation and (d) after oxidation; diagrams showing the electrochemical oxidation currents of (e) the blank solvent and (f) *RRApSVA.

To investigate the CMS quantification method for glycopeptide analysis, a glycosylated peptide, NYIVGQPSS­(β-GlcNAc)­TGNL–OH, was selected for testing. The derivatization yield of this peptide was determined to be 98% by comparing the EIC peak area of the intact peptide (m/z 776.9) signal before and after the derivatization reaction (Table S8). The derivatized peptide was then measured using CMS. Before oxidation, the +1 ion of *NYIVGQPSS­(β-GlcNAc)­TGNL–OH was detected at m/z 858.9 (Figure a), and its isotope peak at m/z 859.9 was used for calculation of the oxidation yield. Upon peptide oxidation, the intensity at m/z 859.9 decreased by 5.3% after oxidation (Figure d) compared to no oxidation (Figure c), indicating a peptide oxidation yield of 5.3%. The total charge Q involved in the oxidation in this case was determined to be 5.2 × 10–8C (Figure e,f). This Q value, along with the oxidation yield, was utilized to calculate the quantity of the derivatized peptide, which was 5.13 pmol. Considering the 98% derivatization yield, the total average peptide amount was calculated to be 5.25 pmol (Table S8). Once again, this value aligns well with the theoretical peptide amount of 5 pmol, with a small quantitation error of 5.0%.

4.

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Mass spectra of *NYIVGQPSS­(β-GlcNAc)­TGNL–OH (a) before electrochemical oxidation at 0.7 V and (b) after electrochemical oxidation; M represents *NYIVGQPSS­(β-GlcNAc)­TGNL–OH. EIC peaks of the *NYIVGQPSS­(β-GlcNAc)­TGNL–OH at m/z 859.9 (c) before oxidation and (d) after oxidation; diagrams showing the electrochemical oxidation currents of (e) the blank solvent and (f) *NYIVGQPSS­(β-GlcNAc)­TGNL–OH.

As shown above, several peptides including phosphopeptides and glycopeptides were tested in this study, and the quantitation errors were good and within ±7%. The results are summarized in Table . The results suggest the feasibility and accuracy of our CMS for quantitation.

1. Summary of CMS Quantitation Results for the Measured Peptides.
peptides injected amount (pmol) measured amount (pmol) quantification error (%)
BOC-Lys-OH 5 4.73 –5.4
RGD 5 4.70 –6.0
GGYR 5 4.73 –5.4
TSTEPQpYQPGENL 10 10.66 6.6
RRLIEDAEpYAARG 10 10.03 0.3
RRApSVA 5 5.20 3.4
NYIVGQPSS(β-GlcNAc) TGNL–OH 5 5.25 5.0
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Quantitation of Glycopeptides from IgG

As a demonstration of the application of our CMS approach, we applied it for quantifying glycopeptides of the antibody IgG. MS techniques for antibody quantification in biotherapeutics typically employ a “bottom-up” strategy, in which proteins are enzymatically digested into peptides. However, quantitation of N-glycosylation is a challenging task. The lack of commercially available N-glycosylated standards precludes absolute quantitation, and thus, relative quantitation of glycosylation is performed by the multiple reaction monitoring method where relative comparisons of glycan/glycopeptide signals are taken into account. In our study, using CMS after peptide derivatization, we enabled standard-free quantification of several glycopeptides originating from the heavy chain Fc of the IgG (located on residue N297, see the sequence information on NIST monoclonal antibody (mAb) in the Supporting Information).

A HILIC column was used for the separation of these glycopeptides due to the high affinity of the glycopeptides. Previously, we reported NIST mAb glycopeptide quantitation by CMS, but the direct electrochemical oxidation of those glycopeptides was complicated due to the presence of two oxidizable tyrosine residues in them. As the oxidation of one or both of the tyrosine residues occurred, the CMS quantitation required the assumption that the oxidation product resulting from one tyrosine oxidation has the same ionization efficiency as the oxidation product resulting from the oxidation of two tyrosine residues, making the calculation not straightforward. In this study, we took the protocol to derivatize these glycopeptides with DPDP, so the oxidation at a low oxidation potential of 0.8 V could selectively occur in the catechol tags of the derivatized glycopeptides rather than the tyrosine residues, thus simplifying the quantitation calculation.

In our study, the NIST mAb was subjected to trypsin digestion, from which five glycopeptides (each with glycosylation at the N297 residue of the Fc heavy chain), EEQYNSTYR-G0F, EEQYNSTYR-G1F, EEQYNSTYR-G2F, EEQYNSTYR-G1F­(-GlcNAc), and EEQYNSTYR-G2F­(Hex) (structures shown in Table ) were identified. The glycopeptides were derivatized using DPDP, and the derivatization yields were found to be 93–98% (Table S9). Subsequently, the derivatized glycopeptides were separated by a HILIC column, and their EICs are shown in Figure a. The major glycoforms detected were *EEQYNSTYR-G0F, *EEQYNSTYR-G1F, and *EEQYNSTYR-G2F based on observed EIC peak intensities, while minor glycoforms included *EEQYNSTYR-G1F­(-GlcNAc) and *EEQYNSTYR-G2F­(Hex). It is noteworthy that *EEQYNSTYR-G1F exhibited a split peak at 22.4 min (the third panel of Figure a) due to partial separation of glycan isomers resulting from differences in galactose α-3/6 linkage. Additionally, the glycopeptide *EEQYNSTYR-G1F­(-GlcNAc) eluted at 20.4 min (second panel of Figure a), with an extra peak observed at 22.4 min, most likely due to in-source fragmentation of the *EEQYNSTYR-G1F ion. The electric oxidation current peaks of these derivatized glycopeptides are shown in Figure b.

2. NIST mAb Glycopeptide Quantities Measured by CMS.

graphic file with name tg5c00047_0007.jpg

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a

Structures using the consortium for functional glycomics notation. Symbol representations of glycans: galactose = yellow circles, mannose = green circles, N-acetylglucosamine = blues boxes, and fucose = red triangles.

5.

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(a) EICs of +2 ions of 5 glycopeptides from tryptic digestion of IgG after derivatization. (b) Electric current responses of the derivatized glycopeptides from IgG digest upon electrochemical oxidation.

For CMS quantitation, prior to electrolysis, the doubly charged ion of *EEQYNSTYR-G0F was detected at m/z 1399.5 (Figure S8,a) along with an in-source oxidation product peak at m/z 1398.5, whereas postelectrolysis analysis revealed the increased intensity of the oxidation product at m/z 1398.5 (Figure S8,b). To avoid interference of the isotope peak of the oxidized product to the ion intensity of the intact peptide ion, we chose the EIC area change from m/z 1401.0, the M + 3 peak of m/z 1399.5, for the oxidation yield calculation. The oxidation yield for *EEQYNSTYR-G0F was determined to be 4.3% (Table S9) based on the EIC peak area drop of m/z 1401.0 upon oxidation (Figure S8g,h). The oxidation current was recorded as 2.86 × 10–7C. According to eq , the measured amount of *EEQYNSTYR-G0F was 29.4 pmol (triplicate measurements, Table S9). Considering the 94% derivatization yield for this glycopeptide (Table S9), the amount of EEQYNSTYR-G0F was found to be 31.3 pmol. Similar calculations were performed for *EEQYNSTYR-G1F, *EEQYNSTYR-G2F, *EEQYNSTYR-G1F­(-GlcNAc), and *EEQYNSTYR-G2F­(Hex), yielding the measured quantities of 33.2, 6.2, 0.9, and 1.3 pmol, respectively (see Table S9, Figures S8 and S9). These glycopeptide quantities were also calculated relative to the theoretical total amount of IgG glycopeptides, which was 106 pmol (10 μL of 5.3 μM IgG, totaling 53.0 pmol which would produce 106 pmol glycopeptides in total by considering that one IgG has two symmetrical heavy chain totals) and listed in Table . The summative amount of the five measured glycopeptides was 72.9 pmol, which is in reasonable agreement with the theoretical value of 106 pmol, with a discrepancy of approximately −31.3% (Table ). This discrepancy likely arose from sample losses during sample preparation such as enzymatic digestion or from the presence of additional minor glycopeptides that were not quantified. Nevertheless, our CMS results thus provide an effective estimation of the precursor glycoprotein quantity. Furthermore, our CMS results were compared with data from previous NIST mAb glycosylation studies by Zhao et al. which involved relative quantification of glycosylated peptides based on MS peak areas. As presented in Table , the relative abundances of the three major glycopeptides (*EEQYNSTYR-G0F, *EEQYNSTYR-G1F, and*EEQYNSTYR-G2F) determined by CMS were close to the value reported by Zhao et al., representing (29.5% vs 38.4%), (31.3% vs 34.7%), and (5.9% vs 7.4%) of total antibody abundance, respectively. While the relative abundances determined by CMS and Zhao’s method are similar, our CMS method uniquely provides absolute quantification for each glycopeptide. These findings underscore the effectiveness and potential utility of CMS for precise glycopeptide quantification.

Conclusions

In this study, we demonstrated an absolute quantitation strategy for quantifying phosphopeptides and glycopeptides by using the CMS approach in combination with an electrochemical labeling technique. Standard peptides, phosphopeptides, and glycopeptides were successfully quantified by CMS with good quantitation accuracy. We also quantified glycopeptides from digested IgG. By this approach, we were able to estimate the amount of glycoprotein based on the abundances of surrogate glycopeptides. The highlight of CMS is that it does not require an isotope-labeled standard to achieve absolute quantity, which has the potential to break the bottleneck in many current methods. It can be seen that our method could be also applicable for quantifying peptides or proteins with other types of PTMs such as S-nitrosation, N-acetylation, methylation, or lipidation, which could contribute to quantitative proteomics.

Supplementary Material

tg5c00047_si_001.pdf (914.8KB, pdf)

Acknowledgments

This work was supported by NSF (CHE-2203284), NIH (1R21GM148874-01), and New Jersey Health Foundation grants (PC 23-23 and PC 38-25).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.5c00047.

  • Schematic diagram; electric current and MS spectra; EICs; CID MS/MS spectra; and MS data as well as experimental setup (PDF)

MTAH, YA, BD, TY, AS, and QY performed the investigation and analysis. HC conceived the project idea and directed the research, and HDD guided us on the electrochemistry experiments. MTAH, YA, and HC wrote the manuscript.

The authors declare no competing financial interest.

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