Absolute Quantitation of Tryptophan-Containing Peptides and Amyloid β-Peptide Fragments by Coulometric Mass Spectrometry

Abstract

Isotope-labeled internal standards are routinely used for mass spectrometry (MS)-based absolute quantitation. However, syntheses of isotope-labeled peptides are time-consuming and costly. To tackle this issue, we recently developed a coulometric mass spectrometric (CMS) approach for absolute quantitation without the use of standards, based on the electrochemical oxidation of cysteine or tyrosine-containing peptides followed by mass spectrometric measurement of the oxidation yield. To further expand the utility of this method, herein we present the CMS method for absolute quantitation of peptides based on tryptophan electrochemical oxidation. Several tryptophan-containing peptides, such as WGG, WQPP­RARI, WAGG­DASGE, RTRPL­WVRME, and KVPR­NQDWL, were successfully quantified with a quantification error ranging from −4.5 to +4.3%. Furthermore, this quantitation approach is also applicable to protein, in which protein can be digested and a surrogate peptide can be selected for quantification to reflect the amount of the parent protein, as exemplified by CMS analysis of peptide GITWK from cytochrome c. The CMS result agreed well with the traditional isotope dilution method, with only a small difference of 3.5%. In addition, CMS was used to successfully quantify amyloid beta (Aβ) peptide fragments (up to 28 amino acid residues) based on tyrosine oxidation. The validity of the CMS method for peptide and protein absolute quantitation without using isotope-labeled peptide standards would greatly facilitate proteomics research.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry

“…Previously, we demonstrated that CMS can be used for absolute quantitation of a single protein based on quantitation of its surrogate peptides. , In this study, we explored the possibility for using CMS for quantitation of multiple proteins in a mixture in one run. We reason that this would be straightforward as long as multiple surrogate peptides representing different proteins could be LC-separated.…”

“…As the protein can be enzymatically digested into peptides and the amount of peptide can reflect the quantity of its precursor protein, our CMS can also be used for absolute quantitation of proteins. However, previously, CMS was only used for the quantitation of single protein including mAb. ,, This study explored CMS for multi-protein quantitation in one mixture sample in the same run and demonstrated the potential of CMS for absolute quantitation of low-abundant HCP in the presence of high-abundant NIST mAb 8671, without using standards. In addition, by using CMS, this study presents simultaneous quantitation of native and deamidated peptides as well as the succinimide intermediate from deamidation of Asn 318 residue of NIST 8671 mAb, for the first time.…”

“…Recently, our laboratory developed coulometric mass spectrometry (CMS) for absolute quantitation of small organic molecules, peptides, and large protein molecules without the need of using standards or calibration curves. − As illustrated in Scheme , an electrochemically active peptide (e.g., those containing either tyrosine or tryptophan residue) is subjected to LC separation, electrochemical oxidation, and MS detection. Upon electrochemical oxidation, electron transfer occurs, resulting in an electrical current peak that can be integrated over time to obtain the total charge Q .…”

“…The integrated EIC peak area of m/z 546.26 after oxidation (Figure 1h) was smaller by 11.3% compared to that before oxidation (Figure 1g), suggesting that the oxidation yield for LDQWLCEK was 11.3% (see detailed data in Table S1). According to the calculation equation reported previously 30 for CMS quantitation of W-containing peptides based on Faraday's law using the integrated oxidation current (i.e., Q = 2.29 × 10 −7 C as shown in Figure 1b) and product intensity ratios (see detailed calculations in Table S1, Supporting Information), the amount of the oxidized LDQWLCEK was calculated as 0.86 pmol. In combination of the oxidation yield of 11.3%, the measured amount of LDQWLCEK was 7.6 pmol.…”

Absolute Quantitation of N-Nitrosamines by Coulometric Mass Spectrometry without Using Standards

“…In this study, to eliminate the need for using standards or calibration curves for quantitation, we present a new strategy of using electrochemistry-assisted mass spectrometry, namely coulometric mass spectrometry (CMS). We recently have applied our CMS method − for direct absolute quantitation of organic compounds, peptides, and proteins. As illustrated in Scheme , our quantitation strategy involves the chemical reduction of N -nitrosamine to hydrazine.…”