Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases

Apffel, Alex; Fischer, Steven M.; Goldberg, Gerson; Goodley, Paul C.; Kuhlmann, Frank E.

doi:10.1016/0021-9673(95)00175-m

Cited by 325 publications

(226 citation statements)

References 19 publications

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“…Until recently, perfusion chromatography utilizing highly cross linked polystyrene-devinylbenzene (POROS) resins has often been used for separation and purification of antibodies [17,18]. This technique suffers from limited separation efficiency attributable to high flow rates of 0.5-3.0 mL/min required for separation and signal suppression due to trifluoroacetic acid (TFA)-containing mobile phases (acetonitril/water/TFA) which is not the most popular solvent for electrospray ionization of proteins [19]. Thus, an alternate combination of mobile phase consist-ing of ethanol/propanol/formic acid was employed as Solvent B for RP LC/MS analysis of glycosylated peptides [20].…”

Section: T He Human Monoclonal Immunoglobulin ␥ (Igg)mentioning

confidence: 99%

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Bondarenko

2005

J. Am. Soc. Mass Spectrom.

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For the first time, the characterization of intact 150-kDa monoclonal antibodies (MAbs) using a commercially available three-dimensional ion-trap mass spectrometer (IT-MS) is reported. The IT-MS analysis was performed on-line with reversed-phase high performance liquid chromatography (RP-HPLC) on a POROS column using a nontraditional solvent system of acetonitrile, isopropanol, ethanol, and water in formic acid. The operating parameters of the IT-MS were optimized by extending the mass range to m/z 4000 and elevating the tube lens offset voltage value to around Ϫ100 V. Mass accuracy better than 300 ppm (Ϯ40 Da) has been routinely achieved for these macromolecules. Multiple peaks 162 Da apart due to the hexose variants of the monoclonal IgG antibodies were partially resolved in mass spectra. Several commercial and chimeric antibodies have been investigated in this study. (J Am Soc Mass Spectrom 2005, 16, 307-311)

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Section: T He Human Monoclonal Immunoglobulin ␥ (Igg)mentioning

confidence: 99%

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Bondarenko

2005

J. Am. Soc. Mass Spectrom.

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“…The flow was maintained at 0.2 ml͞min. The effluent was fed to the flow cell of a Hewlett-Packard Series 1100 diode array detector, whose outlet was connected to a mixing tee receiving 0.1 ml͞min glacial acetic acid pumped by an auxiliary pump (17). The mixture then entered the source of a Hewlett-Packard Series 1100 electrospray mass selective detector.…”

Section: Methodsmentioning

confidence: 99%

Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein

Requena

Groth

Legname

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

145

115

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Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29 -231). Using Cu 2؉ ͞ascorbate, we oxidized SHaPrP(29 -231) in vitro. Oxidation was demonstrated by liquid chromatography͞mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29 -101 region, which includes the histidinecontaining octarepeats, is particularly affected by oxidation. Oxidation was time-and copper concentration-dependent and was evident with copper concentrations as low as 1 M. Concomitant with oxidation, SHaPrP(29 -231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37°C with a 6-fold molar excess of Cu 2؉ . These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

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“…TFA is a common ion-pairing reagent that is often used to reduce the interactions of proteins with silica to obtain better resolution. However, it has been known that TFA suppresses mass spectrometry signal [29,30]. Therefore, the effect of using lower levels of TFA was investigated.…”

Section: Comparison Of Different Flow Rates and Mobile Phase Compositmentioning

confidence: 99%

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Liu

Gaza-Bulseco

Chumsae

2009

J. Am. Soc. Mass Spectrom.

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Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly. (J Am Soc Mass Spectrom 2009, 20, 2258 -2264) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry is one of the most indispensable techniques for the characterization of recombinant monoclonal antibodies because most of the modifications that result in heterogeneity and degradation are involved in molecular weight differences [1,2]. Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on...

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Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases

Cited by 325 publications

References 19 publications

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

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