Effects of Anions on the Positive Ion Electrospray Ionization Mass Spectra of Peptides and Proteins

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...

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

“…The collection of charge states observed by ESI mass spectrometry (ESI-MS) for a particular molecule under a given set of experimental conditions is referred to as the charge state distribution (CSD). The charge state distributions of macromolecules can be influenced by many factors, such as the extent of analyte charging, including analyte conformation [3,4], solution pH [5], competition for charge between the analyte and other species [5][6][7][8][9][10], instrumental parameters and desolvation conditions [11], solvent and analyte basicity [8,12], and solvent surface tension [13]. Many of these factors are interrelated.…”

mentioning

confidence: 99%

Basic Vapor Exposure for Tuning the Charge State Distribution of Proteins in Negative Electrospray Ionization: Elucidation of Mechanisms by Fluorescence Spectroscopy

Girod

Antoine

Dugourd

et al. 2012

Manipulation for simplifying or increasing the observed charge state distributions of proteins can be highly desirable in mass spectrometry experiments. In the present work, we implemented a vapor introduction technique to an Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA) source. An apparatus was designed to allow for the enrichment of the nitrogen sheath gas with basic vapors. An optical setup, using laser-induced fluorescence and a pH-chromic dye, permits the pH profiling of the droplets as they evaporate in the electrospray plume. Mechanisms of pH droplet modification and its effect on the protein charging phenomenon are elucidated. An important finding is that the enrichment with basic vapors of the nitrogen sheath gas, which surrounds the nebulizer spray, leads to an increase in the spray current. This is attributed to an increase in the electrical conductivity of water-amine enriched solvent at the tip exit. Here, the increased current results in a generation of additional electrolytically produced OH -ions and a corresponding increase in the pH at the tip exit. Along the electrospray plume, the pH of the droplets increases due to both droplet evaporation and exposure to basic vapors from the seeded sheath gas. The pH evolution in the ESI plume obtained using pure and basic seeded sheath gas was correlated with the evolution of the charge state distribution observed in mass spectra of proteins, in the negative ion mode. Taking advantage of the Agilent Jet Stream source geometry, similar protein charge state distributions and ion intensities obtained with basic initial solutions, can be obtained using native solution conditions by seeding the heated sheath gas with basic vapors.

“…A relatively high CSD, similar to that of Figure 3b, was generated along with a lower CSD centered at +7. Cytochrome c is known to fold into a molten globule state [70][71][72][73][74][75]. However, the bimodal distribution observed in Figure 3d has not been observed for cytochrome c prepared at any solution pH.…”

Section: Cytochrome C and Ubiquitin: Positive Polaritymentioning

confidence: 90%

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

Kharlamova

DeMuth

McLuckey

2011

The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK a or pK b of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry.