Nanoelectrospray Ionization of Protein Mixtures: Solution pH and Protein p I

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Multiply protonated human hemoglobin ␣-chain species, ranging from [M ϩ 4H] 4ϩ to [M ϩ 20H] 20ϩ , have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D 75 /M 76 , also noted previously in beam-type collisional activation studies, was observed for the [M ϩ 8H] 8ϩ to [M ϩ 11H] 11ϩ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D 75 /M 76 channel. The [M ϩ 12H] 12ϩ -[M ϩ 16H] 16ϩ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M ϩ 4H] 4ϩ ion, all charge states showed cleavage at the L 113 /P 114 bond. This cleavage proved to be the most prominent dissociation for charge states [M ϩ 14H] 14ϩ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach. (J Am Soc Mass Spectrom 2006, 17, 923-931)

Section: Resultsmentioning

confidence: 96%

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Mekecha

Amunugama

McLuckey

2006

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mentioning

confidence: 99%

“…McLuckey and coworkers [21] measured the signal intensities (related directly to the ionization efficiency) of analyte ions from protein mixtures electrosprayed at various pH values and deter-mined that the main parameter influencing ionization efficiency was the protein surface charge. Likewise, Kuprowski and Konermann [22] showed that the signal intensity of ions from ESI of denatured proteins with higher surface charge is greater than the signal intensity of ions from the ESI native state proteins.…”

mentioning

confidence: 99%

Monte carlo simulation of macromolecular ionization by nanoelectrospray

Hogan

Biswas

2008

Electrospray ionization (ESI) is commonly used in macromolecular mass spectrometry, yet the dynamics of macromolecules in ESI droplets are not well understood. In this study, a Monte Carlo based model was developed, which can predict the efficiency of electrospray ionization for macromolecules, i.e., the number of macromolecular ions produced per macromolecules electrosprayed. The model takes into account ESI droplet evaporation, macromolecular diffusion within the droplet, droplet fissions, and the statistical nature of the ESI process. Two idealized representations of macromolecular analytes were developed, describing cluster prone, droplet surface inactive macromolecules and droplet surface active macromolecules, respectively. It was found that surface active macromolecules are preferentially ionized over surface inactive cluster prone macromolecules when the initial droplet size is large and the analyte concentration in solution is high. Simulations showed that ESI efficiency decreases with increasing initial droplet size and analyte molecular weight, and is influenced by analyte surface activity, the properties of the solvent, and the variance of the droplet size distribution. Model predictions are qualitatively supported by experimental measurements of macromolecular electrospray ionization made previously. Overall, this study demonstrates the potential capabilities of Monte Carlo based ESI models. Future developments in such models will allow for more accurate predictions of macromolecular ESI intensity. (J Am Soc Mass Spectrom 2008, 19, 1098 -1107) © 2008 American Society for Mass Spectrometry E lectrospray ionization (ESI) allows for the production of gas-phase ions from nonvolatile species in solution, and is particularly useful in the measurement of biological macromolecules with masses in the kDa and MDa ranges. Despite its widespread use, many aspects of the ESI of macromolecules are poorly understood [1]. The efficiency of the ESI process, i.e., the number of gas-phase ions produced per number of analytes electrosprayed, is a fundamental parameter in ESI that is typically unknown [2]. Droplets of the electrosprayed solution are produced in ESI, which have multiple excess charges on their surface [3]. Analytes are ionized from these droplets by one of two mechanisms: the charged residue mechanism [4] or the ion emission mechanism [5,6]. Analyte ionization by the charge residue mechanism requires that analytes be separated from one and other (one analyte per droplet) [7]. This is accomplished by Coulombic fissions of evaporating charged droplets in which unstable droplets emit smaller, charged progeny droplets [8]. Although complete separation of analytes is not necessary in the ion emission mechanism, ESI droplets must have diameters about 10 nm for ion emission to occur [6], and ion emission also occurs only after a series of droplet fission events [9].The parameters governing the ionization of small analytes, which are believed to be ionized by the ion emission mechanism [10], have been examined e...

“…Their net charge in solution is negative, resulting in significantly lower ion yields using ESI of the opposite polarity [63]. However, ECD of doubly-protonated nucleic acids [64] or CAD of multiply-charged protein anions [7] can provide useful structural information.…”

Section: Methods For Dissociation Of Multiply-charged Biomolecular Ionsmentioning

confidence: 99%

Top-down identification and characterization of biomolecules by mass spectrometry

Breuker

Jin

Han

et al. 2008

112

The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10 5 ) mass resolving power and accuracy (~1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and posttranslational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation". The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.