Effects of alkali on proteins. Disulfides and their products

Nashef, Aws S.; Osuga, David T.; Lee, Honson S.; Ahmed, Ahmed I.; Whitaker, John R.; Feeney, Robert E.

doi:10.1021/jf60210a020

Cited by 158 publications

(93 citation statements)

References 34 publications

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“…The mass spectra of peaks a to c are shown in Figure 4b. The theoretical molecular weights of antibody-A heavy chain without C-terminal Lys and with a core-fucosylated biantennary complex oligosaccharide structure without terminal galactose ( linkage has been well documented in the literature [31][32][33] and reported for monoclonal antibodies [17,28]. No interpretable mass spectrum was obtained for the shoulder that eluted in front of peak a.…”

Section: On-line Sec-msmentioning

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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Section: On-line Sec-msmentioning

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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“…The reduction in FAN in the Type II tannin sorghum with NaOH steeping may be due to denaturation of protein by the NaOH. Alkali treatment of protein results in losses of some amino acids and alteration of others (Nashef, Osuga, Lee, Ahmed, Whitaker & Feeney, 1977). Such reactions can alter protein structure, thereby limiting it susceptibility to enzymic hydrolysis.…”

Section: Effects Of Naoh Steeping Of Sorghum Flour On Brewing/bioethamentioning

confidence: 99%

Inactivation of tannins in milled sorghum grain through steeping in dilute NaOH solution

2015

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.Running title: Tannin inactivation in milled sorghum grain with dilute NaOH 1 Highlights  Steeping milled Type III tannin sorghum in NaOH solution reduces tannin content  NaOH treatment substantially reduces α-amylase inhibition by the tannins  Tannin polymerization was found to be the mechanism of inactivation  NaOH treatment could enable use of Type III tannin sorghum in bioethanol production 2 ABSTRACTSteeping milled sorghum in up to 0.4% NaOH was investigated as a method of tannin inactivation. NaOH steeping substantially reduced assayable total phenols and tannins in both Type III and Type II sorghums and with Type III sorghum caused a 60-80% reduction in α-amylase inhibition compared to a 20% reduction by water steeping. NaOH treatment also reduced starch liquefaction time and increased free amino nitrogen. Type II tannin sorghum did not inhibit α-amylase and consequently the NaOH treatment had no effect. HPLC and LC-MS of the tannin extracts indicated a general trend of increasing proanthocyanin/procyanidin size with increasing NaOH concentration and steeping time, coupled with a reduction in total area of peaks resolved. These show that the NaOH treatment forms highly polymerised tannin compounds, too large to assay and to interact with the α-amylase. NaOH pre-treatment of Type III sorghums could enable their utilization in bioethanol production.

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“…The proposed fragmentation pathways for the formation of dehydroalanine, thioaldehyde, and reduced glutathione are reminiscent of chemical cleavages occurring at the cystine site in the peptide under base catalyzed condition in solutions [23][24][25]. We have attempted to obtain experimental support for these proton abstraction processes by subjecting a deuterated sample of glutathione, in which all exchangeable hydrogens have been replaced by deuterium, to collision induced dissociation in an ion trap mass spectrometer.…”

Section: Contryphanmentioning

confidence: 99%

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Thakur

Balaram

2008

J. Am. Soc. Mass Spectrom.

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The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of C ␣ H and C ␤ p H protons resulting in product ions derived by neutral loss of H 2 S 2 and H 2 S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products. Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7]. The use of metal ions as gas-phase disulfide cleavage agents has also been investigated [12]. In general, only limited information regarding structure can be derived from intact disulfide peptides in the positive ion mode. In contrast, several recent studies have emphasized the utility of negative ion mass spectrometry [7,10,[13][14][15][16][17][18]. Specifically, Bowie and coworkers have presented assignments of product ion spectra obtained by negative ion electrospray mass spectrometry of peptides containing both intra and inter molecular disulfide bond [10,18]. The present study complements the work of Bowie and coworkers and further provides mechanistic details by examining second generation product ions by using ion trap mass spectrometry.In the structural analysis of peptides containing intact disulfides, under negative ion conditions, proton abstraction from the C ␣ H and C ␤ H positions of the Cys residue results in diverse fragmentation reactions, leading to cleavage of disulfide linkages. In the case of peptides that contain labile peptide bonds, for example X-Pro sequences, preferential cleavage at these sites results in two linear chains linked by a disulfide bond, which then undergo further f...

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Effects of alkali on proteins. Disulfides and their products

Cited by 158 publications

References 34 publications

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

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

Inactivation of tannins in milled sorghum grain through steeping in dilute NaOH solution

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

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