Supercharged Protein and Peptide Ions Formed by Electrospray Ionization

122

Protein ions, after mass spectrometric separation, can be soft-landed into liquid surfaces with preservation of their native structures. Retention of biological activity is strongly favored in glycerol-based surfaces but not in self-assembled monolayer solid surfaces. Soft-landing efficiency for multiply-charged hexokinase ions was found to be some four times higher for a glycerol/fructose liquid surface than for a fluorinated self-assembled monolayer surface. Soft-landing into liquid surfaces is also shown to allow (1) protein purification, (2) on-surface identification of the soft-landed material using MALDI, and (3) protein identification by in-surface tryptic digestion. Pure lysozyme was successfully isolated from different mixtures including an oxidized, partially decomposed batch of the protein and a partial tryptic digest. Liquid glycerol/carbohydrate mixtures could be used directly to record MALDI spectra on the soft-landed compounds provided they were fortified in advance with traditional MALDI matrices such as p-nitroaniline and ␣-cyano-4-hydroxycinnamic acid. Various proteins were soft-landed and detected on-target using these types of liquid surface. Soft-landing of multiply-charged lysozyme ions onto fluorinated self-assembled monolayer surfaces was found to occur with a limited amount of neutralization, and trapped multiply-charged ions could be desorbed from the surface by laser desorption. Initial data is shown for a new approach to protein identification that combines top-down and bottom-up approaches by utilizing protein ion soft-landing from a protein mixture, followed by tryptic digestion of the landed material and detection of characteristic tryptic fragments by MALDI. , has been applied in experiments ranging from studies using chemically inert and structurally organized self-assembled monolayers (SAMs) as substrates for ion storage [2], to studies of ion mobility through ice and vapor deposited films [3,4], to experiments aimed at developing a separation method in a form of preparatory mass spectrometry [5,6]. Simple organic cations [7,8] [12][13][14] have all been the targets of examination by ion soft-landing. The separation, purification, and storage of chemical species by a completely non-destructive, mass spectrometric approach are the objectives of the studies currently underway in our laboratory.In a preliminary report [5], we described a method which showed the potential for generating biologically active protein chips from protein mixtures by massselective ion soft-landing. After ionization of the mixture by electrospray (ESI), individual protein ions were mass-selected and soft-landed at predetermined locations on the surface. The results showed that the use of mass spectrometry as a separation method in biology provides high selectivity because components with different molecular formulas can be separated and softlanded. However, retention of the biological activity of a protein or other biomolecule in the course of this (or any mass spectrometry experiment) is a much more chall...

Section: Isolation Of Proteins From Complex Mixtures By Ion Soft-landingmentioning

confidence: 99%

Ion soft-landing into liquids: Protein identification, separation, and purification with retention of biological activity

Gologan

Takáts

Alvarez

et al. 2004

122

“…Iavarone et al was the pioneer in studies of supercharging [12]. They observed an increase in the charge state for peptides if additives such as diethylamine, 2-methoxyethanol, ethylene glycol, glycerol, or m-nitrobenzyl alcohol were added to the solution [12][13][14]. Although the mechanism of supercharging is yet to be fully explained, the authors related this effect with the additives being less volatile than water and thus increasing surface tension.…”

Section: Introductionmentioning

confidence: 99%

Ionization Efficiency of Doubly Charged Ions Formed from Polyprotic Acids in Electrospray Negative Mode

Liigand

Kaupmees

Kruve

2016

Abstract. The ability of polyprotic acids to give doubly charged ions in negative mode electrospray was studied and related to physicochemical properties of the acids via linear discriminant analysis (LDA). It was discovered that the compound has to be strongly acidic (low pK a1 and pK a2 ) and to have high hydrophobicity (logP ow ) to become multiply charged. Ability to give multiply charged ions in ESI/MS cannot be directly predicted from the solution phase acidities. Therefore, for the first time, a quantitative model to predict the charge state of the analyte in ESI/MS is proposed and validated for small anions. Also, a model to predict ionization efficiencies of these analytes was developed. Results indicate that acidity of the analyte, its octanol-water partition coefficient, and charge delocalization are important factors that influence ionization efficiencies as well as charge states of the analytes. The pH of the solvent was also found to be an important factor influencing the ionization efficiency of doubly charged ions.

“…More highly charged peptides also have increased coulombic repulsion, which may facilitate dissociation compared with lower-charged species. Iavarone and Williams [35][36][37][38][39] explored the use of additives, such as m-nitrobenzyl alcohol (m-NBA), that increase the surface tension and reduce the vapor pressure of electrospray ionization (ESI) droplets, thus generating higher protein and peptide charge states. This phenomenon has been termed supercharging.…”

mentioning

confidence: 99%

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

Madsen

Brodbelt

2009

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c. , but uninformative labile neutral losses for both unmodified and modified peptides remain a consistent problem. Recently, electron capture dissociation (ECD) [2] in Fourier transform ion cyclotron resonance (FT-ICR) instruments, a method that involves the reaction of multiply charged cations with low-energy electrons, and electron-transfer dissociation (ETD) [3] in ion trap instruments, a technique that exploits the reaction of multiply charged cations with radical anions, have been developed to combat this neutral loss problem by affording complementary c-and z-type backbone cleavages that leave labile modifications intact. These methods have been particularly promising for characterization of PTMs [4 -8]; however, both techniques suffer from low fragmentation efficiencies, especially for lower charge states [9, 10], compared with CID.Infrared multiphoton dissociation (IRMPD) is another tandem MS (MS/MS) method that has been successfully implemented in FT-ICR, time-of-flight, and quadrupole ion trap instrument systems [6,[11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Much of the appeal of IRMPD in quadrupole ion traps is related to the broader m/z trapping range possible than that for conventional CID. This stems from the ability to reduce the radio frequency (rf) trapping voltage during ion activation, an option that is detrimental for CID as a result of the decrease in energy deposition associated with lower rf trapping voltages [25]. This low-mass cutoff (LMCO) problem associated with CID prohibits the detection of many diagnostic b and y ions of lower m/z as well as immonium ions, the latter of which are particularly useful in determining the amino acid composition of unknown peptides. Furthermore, these lower m/z diagnostic ions are important for de novo sequencing algorithms and can be useful for the rapid determination of modified...