Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Infrared Multiple Photon Dissociation Action Spectroscopy and Theoretical Studies of Diethyl Phosphate Complexes: Effects of Protonation and Sodium Cationization on Structure

The gas-phase structures of deprotonated, protonated, and sodium-cationized complexes of diethyl phosphate (DEP) including [DEP − H]⁻, [DEP + H]⁺, [DEP + Na]⁺, and [DEP − H + 2Na]⁺ are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy using tunable IR radiation generated by a free electron laser, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) with an electrospray ionization (ESI) source, and theoretical electronic structure calculations. Measured IRMPD spectra are compared to linear IR spectra calculated at the B3LYP/6-31G(d,p) level of theory to identify the structures accessed in the experimental studies. For comparison, theoretical studies of neutral complexes are also performed. These experiments and calculations suggest that specific geometric changes occur upon the binding of protons and/or sodium cations, including changes correlating to nucleic acid backbone geometry, specifically P–O bond lengths and ∠OPO bond angles. Information from these observations may be used to gain insight into the structures of more complex systems, such as nucleotides and solvated nucleic acids.     

Atmospheric pressure ionization mass spectrometry techniques for the analysis of alkyl ethoxysulfate mixtures

This paper compares two liquid introduction atmospheric pressure ionization techniques for the analysis of alkyl ethoxysulfate (AES) anionic surfactant mixtures by mass spectrometry, i. e., electrospray ionization (ESI) in both positive and negative ion modes and atmospheric pressure chemical ionization (APCI) in positive ion mode, using a triple quadrupole mass spectrometer. Two ions are observed in ESI(+) for each individual AES component, [M + Na]⁺ and a “desulfated” ion [M − SO₃ + H]⁺, whereas only one ion, [M − Na]⁻ is observed for each AES component in ESI(−). APCI(+) produces a protonated, “desulfated” ion of the form [M − NaSO₃ + 2H]⁺ for each AES species in the mixture under low cone voltage (10 V) conditions. The mass spectral ion intensities of the individual AES components in either the series from ESI(+) or APCI(+) can be used to obtain an estimate of their relative concentrations in the mixture and of the average ethoxylate (EO) number of the sample. The precursor ions produced by either ESI(+) or ESI(−), when subjected to low-energy (50 eV) collision-induced dissociation, do not fragment to give ions that provide much structural information. The protonated, desulfated ions produced by APCI(+) form fragment ions which reveal structural information about the precursor ions, including alkyl chain length and EO number, under similar conditions. APCI(+) is less susceptible to matrix effects for quantitative work than ESI(+). Thus APCI(+) provides an additional tool for the analysis of anionic surfactants such as AES, especially in complex mixtures where tandem mass spectrometry is required for the identification of the individual components.     

Effects of disulfide linkages on gas-phase reactions of small multiply charged peptide ions

Multiply protonated ions of disulfide-intact and -reduced peptides were generated by electrospray ionization and studied by Fourier transform ion cyclotron resonance mass spectrometry. The effects of disulfide bonds on gas-phase deprotonation reactions and hydrogen/deuterium (H/D) exchange were investigated. Insight into conformations was gained from molecular dynamics calculations. For ions from three small peptides containing 9–14 amino acid residues, H/D exchange is more sensitive to changes in conformation than deprotonation. However, with both gas-phase reactions the more diffuse forms of the peptides (as determined by molecular modeling) react more readily. The effects of disulfide cleavage on the conformations and on the reactions were found to depend upon the sequence of the peptide. For [M + 3H]³⁺ of TGF-α (34–43), reduction of the disulfide linkage leads to a greatly extended structure and a dramatic increase in the rate and extent of H/D exchange. In contrast, [M + 2H]²⁺ of Arg⁸ -vasopressin becomes slightly more compact upon cleavage of the disulfide bond; these reduced ions are slower to react. For [M + 3H]³⁺ of somatostatin-14, reduction of the disulfide bond has little effect on conformation or gas-phase reactivity. Overall, these results indicate that there is no general rule on how cleavage of a disulfide bond will effect a peptide ion’s gas-phase reactivity.     

H/D exchange kinetics: Experimental evidence for formation of different b fragment ion conformers/isomers during the gas-phase peptide sequencing

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with H/D exchange reactions was utilized to explore the existence of different b₅⁺ and b₄⁺ fragment ion conformers/isomers of hexapeptide WHWLQL in the gas phase. Distinct H/D exchange trends for protonated WHWLQL ([M + H]⁺) and its b₅⁺ and b₄⁺ fragment ions (with ND₃) were observed. Isolated ¹²C_all isotopomers of both b₅⁺ and b₄⁺ fragment ions yielded bimodal distributions of H/D exchanged product ions. The H/D exchange reaction kinetics also confirmed that b₅⁺ and b₄⁺ fragment ions exist as combination of slow-exchanging (“s”) and fast-exchanging (“f”) species. The calculated rate constant for the first labile hydrogen exchange of [M + H]⁺ (k_{[M + H]} ⁺ = 3.80 ± 0.7 × 10⁻¹⁰ cm³ mol⁻¹ s⁻¹) was ∼30 and ∼5 times greater than those for the “s” and “f” species of b₅⁺, respectively. Data from H/D exchange of isolated “s” species at longer ND₃ reaction times confirmed the existence of different conformers or isomers for b₅⁺ fragment ions. The sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of WHWLQL combined with the H/D exchange reactions indicate that “s” and “f” species of b₅⁺ and b₄⁺ fragment ions can be produced in the ICR cell as well as the ESI source. The significance of these observations for detailed understanding of protein sequencing and ion fragmentation pathways is discussed.     

Effect of electrospray ionization conditions on low-energy tandem mass spectra of peptides

The effect of electrospray ionization (ESI) conditions on low-energy tandem mass spectra of peptides in the relative molecular mass range 400–1200 was examined. For singly charged peptide ions the source skimmer potential (which determines the degree of acceleration of the ions through the intermediate pressure region in the source) can strongly influence the extent of fragmentation observed in tandem mass spectra, especially at low collision energies. For each peptide there is an optimum skimmer potential which represents a balance between generating ions with sufficient internal energy for subsequent tandem mass spectrometric experiments and inducing the onset of other processes such as source fragmentation. The fragmentation which can be achieved in tandem mass spectra with high skimmer potentials differs from ESI source fragmentation for the same peptides. We have found that fragmentation in ESI mass spectra depends both on skimmer potential and on solvent pH, presumably because the latter determines the proportion of doubly charged species generated from a given peptide. Low-energy tandem mass spectra of peptides following ESI are equally as sensitive to peptide structure and the type of adduct studied (e.g. [M + H]⁺ vs. [M + NH₄]⁺) as tandem mass spectra obtained following older ionization methods such as fast atom bombardment.     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.     

Quantitative aspects of analyzing small molecules – monitoring singly or doubly charged ions? A case study of ximelagatran

Precision, reproducibility and lower limit of quantitation (LLOQ) are important characteristics of a quantitative method. We have investigated these properties for Ximelagatran (Xi), which has a high tendency to form doubly charged ions in electrospray ionization (ESI), by studying the percentage of doubly charged species formed when varying the formic acid (FA) concentration, analyte concentration, amount of organic modifier and flow rate. It was found that the percentage of [Xi + 2H]²⁺ can be controlled to be more than 90% or less than 10% by varying the amount of FA present, and that the change between these values is dramatic. Furthermore, the percentage of [Xi + 2H]²⁺ formed decreases with increased analyte concentration and increased flow rate. No apparent relationship with the amount of organic modifier was found. The results have the implication that, by carefully controlling the selected parameters, the LLOQ, precision and reproducibility can be improved. We have compared the fragmentation of the singly and doubly charged species and concluded that the [Xi + 2H]²⁺ ion is more inclined to undergo fragmentation than [Xi + H]⁺. As a consequence, unusual instrumental settings had to be used for the experiments. The fragmentation patterns are to a great extent similar, but the doubly charged species is more inclined to generate low‐mass product ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of liquid chromatography–tandem mass spectrometry for the characterization of galactosylated and tagatosylated β-lactoglobulin peptides derived from in vitro gastrointestinal digestion

This article describes a comprehensive characterization of bovine β-lactoglobulin peptides glycated with an aldohexose (galactose) or a ketohexose (tagatose), derived from in vitro gastrointestinal digestion, by liquid chromatography coupled to positive electrospray ion trap tandem mass spectrometry. In addition to the dissociation pathway previously described for aldohexoses-derived Amadori compounds, i.e. formation of the pyrylium ([M+H]⁺-54) and furylium ions ([M+H]⁺-84) via the oxonium ion ([M+H]⁺-18), another and more direct fragmentation route involving the formation of the imminium ion ([M+H]⁺-150) is also reported following extensive glycation rates of β-lactoglobulin with both carbohydrates. These results indicated that the analysis of digested proteins by LC-ESI-MS² on a three-dimensional ion trap monitoring neutral losses is an efficient and direct method to identify peptides glycated not only through the Amadori rearrangement but also via the Heyns rearrangement. Nevertheless, as the predominating MS² fragmentation pattern of the glycated peptides is derived from the sugar moiety, the sequence-informative b- and y-ions resulting from peptide backbone cleavage were undetected. To overcome this drawback, and taking advantage of multi-stage fragmentation capabilities of ion traps, the indicative Amadori and Heyns-derived imminium ions were successfully used in MS³ analyses to identify the peptide backbone, as well as the specific glycation site. In addition, further MS⁴ analyses were needed to carry out the characterization of doubly glycated peptides.     

Hydralazine derivative of aldehyde: A new type of [M − H]+ ion formed in electrospray ionization mass spectrometry

Hydralazine has been widely employed in the development of drugs, derivatization reagents, and ligands. In the present work, we reported a new type of dehydrogenated ion [M ? H]⁺ that was produced from the hydralazine derivative of hexanal in electrospray ionization mass spectrometry (ESI‐MS). The formation of [M ? H]⁺ ions in the ESI‐MS was found to be independent on the mobile phase composition of the liquid chromatography and ESI source parameters. A series of hydralazine derivatives of aldehyde were investigated to confirm this phenomenon. The results showed that hydralazine derivatives of aldehydes that contained an sp³ hybridization carbon with a hydrogen at the α‐position of aldehydes could form the unexpected [M ? H]⁺ ions, whereas hydralazine derivative of acetone could only generate [M + H]⁺ ion in the ESI‐MS. We proposed the possible formation mechanism of [M ? H]⁺ ion for the hydralazine derivatives of aldehydes: the [M ? H]⁺ ion was possibly formed by the loss a hydrogen molecule (H₂) from the protonated ion [M + H]⁺. The results obtained from density functional theory (DFT) calculations supported this proposed formation mechanism of [M ? H]⁺ ion.     

Entrapment of amino acids in gas phase surfactant assemblies: The case of tryptophan confined in positively charged (1R,2S)‐dodecyl (2‐hydroxy‐1‐methyl‐2‐phenylethyl) dimethylammonium bromide aggregates

The ability of positively charged aggregates of the surfactant (1R ,2S )‐dodecyl(2‐hydroxy‐1‐methyl‐2‐phenylethyl)dimethylammonium bromide (DMEB) to incorporate D‐tryptophan or L‐tryptophan in the gas phase has been investigated by electrospray ion mobility mass spectrometry (ESI‐IM‐MS). Strongly impacted by the pH of the electrosprayed solutions, both protonated (T⁺) and deprotonated (T⁻) tryptophan are effectively included into the aggregates, whereas, tryptophan in zwitterionic (T⁰) form is practically absent in singly charged DMEB aggregates but can be found in multiply charged ones. The ability to incorporate tryptophan increases with the aggregation number and charge state of aggregates. More than 1 tryptophan species can be entrapped (aggregates including up to 5 tryptophan are observed). Collision induced dissociation experiments performed on the positively singly charged DMEB hexamer containing 1 T⁻ show that at low collision energies the loss of a DMEB molecule is preferred with respect to the loss of the DMEB cation plus T⁻ species which, in turn, is preferred with respect to the loss of mere tryptophan, suggesting that the deprotonated amino acid is preferentially located in proximity of a DMEB head group and with the ionic moiety pointing towards the core of the aggregate. The analysis of the collision cross sections (CCS) of bare and tryptophan containing aggregates allowed evaluating the contributions of tryptophan and bromide ions to the total aggregate CCS. No significant discrimination between D‐tryptophan and L‐tryptophan by the chiral DMEB aggregates has been evidenced by mass spectra data, CID experiments, and CCS values.     

Gas‐phase fragmentation of γ‐lactone derivatives by electrospray ionization tandem mass spectrometry

Fragmentation reactions of β‐hydroxymethyl‐, β‐acetoxymethyl‐ and β‐benzyloxymethyl‐butenolides and the corresponding γ‐butyrolactones were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) using collision‐induced dissociation (CID). This study revealed that loss of H₂O [M + H ?18]⁺ is the main fragmentation process for β‐hydroxymethylbutenolide (1) and β‐hydroxymethyl‐γ‐butyrolactone (2). Loss of ketene ([M + H ?42]⁺) is the major fragmentation process for protonated β‐acetoxymethyl‐γ‐butyrolactone (4), but not for β‐acetoxymethylbutenolide (3). The benzyl cation (m/z 91) is the major ion in the ESI‐MS/MS spectra of β‐benzyloxymethylbutenolide (5) and β‐benzyloxymethyl‐γ‐butyrolactone (6). The different side chain at the β‐position and the double bond presence afforded some product ions that can be important for the structural identification of each compound. The energetic aspects involved in the protonation and gas‐phase fragmentation processes were interpreted on the basis of thermochemical data obtained by computational quantum chemistry. Copyright © 2009 John Wiley & Sons, Ltd.     

The reactivity of ferrocenylalkyl azoles under the conditions of electrospray ionization

Under the conditions of electrospray ionization of ferrocenylalkyl azoles FcCH(R)X (Fc-η⁵-C₅H₅Fe-η⁵-C₅H₄, R - H, Me, XH - 2-methyl imidazole, pirazole) the processes of oxidation, protonation, fragmentation and ferrocenylalkylation to form, molecular ions [М]⁺, protonated molecules [М+Н]⁺, ferrocenylalkyl cations [FсCHR]⁺ and bisferrocenylalkyl azole cations [(FcCHR)2X]⁺, respectively, take place. Using special experimental techniques (deuterated solvents, saturation of ionic source of an ESI mass-spectrometer by the vapors of solvents, the experiments under the “inverse” ESI conditions when the solvent is subjected to electrospray in the presence of ferrocenylalkyl derivative vapours) and quantum-chemical calculations at the level of the B3LYP/LanL2DZ theory the scheme of the formation of these ions in a gas phase according to the mechanism of “activating protonation” was suggested. it was found that all these ions are formed through the protonation stage, which is taking place mainly in a gas phase. The key stage is the exothermic process of the protonation of the initial compounds by hydroxonium ions giving rise to protonated [M+H]⁺ molecules which further oxidize and alkylate ferrocenylalkylazoles to form molecular radical cations and bisferrocenylalkyl azole ions [FcCH(Me)-X-CH(Me)Fc]⁺. The decomposition of protonated ions with the elimination of the azole molecule gives rise to ferrocenylalkyl cations [FсCHR]⁺ capable in turn of oxidizing and alkylating the initial compounds.     

Characterization of the Retro-[3+2] Reaction of Protonated 2,3′-Bisindolylmethanes by Electrospray Ionization–Tandem Mass Spectrometry

Twelve 2,3′-bisindolylmethanes with various substituents were investigated using electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. A retro-[3+2] reaction was observed in the collision-induced dissociation spectra of protonated 2,3′-bisindolylmethanes for the first time. The mechanism of retro-[3+2] reaction was concerted or stepwise. For the concerted pathway, carbon–carbon bonds of a protonated compound simultaneously cracked and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) was observed with hydrogen–deuterium exchange labeling. The stepwise pathway goes through 1,3-hydrogen migration twice and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) and m/z 207 ion ([C₁₅H₁₁DN]⁺) were detected with deuterium labeling. In the deuterium-labeled tandem mass spectrum for one compound, only the peak at m/z 208 was present at high abundance, suggesting that the concerted pathway is more likely. In addition, the substituents have no obvious trends on the ratios of the product intensity to the base intensity, further supporting the concerted pathway.     

Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides

Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.     

Tandem mass spectrometry based investigation of cinnamylideneacetophenone derivatives: valuable tool for the differentiation of positional isomers

Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24 derivatives were systematically studied by tandem mass spectrometry (MS²) with electrospray ionization (ESI), in positive ion mode. The protonated molecules, [M + H]⁺, formed under ESI conditions were induced to dissociate and the fragmentation patterns were studied. The information collected provided important structural information on the type of substituents present and constitute an important database concerning this family of compounds. Overall, it was found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI‐MS² fragmentation considerably. These results indicate that ESI‐MS² is a useful technique for distinguishing positional isomers of these cinnamylideneacetophenone derivatives. Copyright © 2011 John Wiley & Sons, Ltd.     

Are liquid chromatography/electrospray tandem quadrupole fragmentation ratios unequivocal confirmation criteria?

Multiple reaction monitoring (MRM) ratios as provided by tandem mass spectrometers are used to confirm positive residue findings (e.g. veterinary drugs or pesticides). The Commission Decision 2002/657/EEC defines tolerance levels for MRM ratios, which are intended to prevent the reporting of false positives. This paper reports findings where blank sample extracts have been spiked by a drug (difloxacin) and the corresponding measured MRM ratios significantly deviated from MRM ratios observed in matrix‐free solution. The observation was explained by the formation of two different [M+H]⁺ analyte ions within the electrospray ionization (ESI) interface. These two ions vary only by the site of analyte protonation. Since they are isobaric, they are equally transmitted through the first quadrupole, but are differently fragmented in the collision chamber. The existence of two isobaric ions was deduced by statistical data and the observation of a doubly charged analyte ion. It was hypothesized that the combined presence of [M+H]⁺ and [M+2H]²⁺ implies the existence of two different singly charged ion species differing only by the site of protonation. Low‐ and high‐energy interface‐induced fragmentation was performed on the samples. The surviving precursor ion population was mass selected and again fragmented in the collision chamber. Equal product ion spectra would be expected. However, very different product ion spectra were observed for the two interface regimes. This is consistent with the assumption that the two postulated isobaric precursor ions show different stability in the interface. Hence the abundance ratio among the two types of surviving precursor ions will shift and change the resulting product ion spectra. The existence of the postulated singly charged ions with multiple chargeable sites was finally confirmed by successful ion mobility separation. Copyright © 2009 John Wiley & Sons, Ltd.     

Reduction of chemical noise in electrospray ionization mass spectrometry by supplemental IR activation

Supplemental infrared (IR) activation was applied to reduce background chemical noise and increase analyte ion signal in a linear ion trap mass spectrometer. Peptides, proteins, and small molecules were all introduced by electrospray ionization, and when regions of chemical noise were isolated and subjected to IR irradiation, protonated analyte molecules were observed in the product ion mass spectra. By isolating the entire mass range (e.g., m/z 400–2000) and then irradiating all ions in the trap, supplemental IR activation increased the signal of singly protonated peptides by almost 70% and by 40%–55% for the lower charge states of cytochrome c. This increase in analyte ion signal was less dramatic for the higher charge states of peptides and proteins. The chemical noise present in the mass spectra is attributed to incomplete desolvation of the electrospray, as the abundance of the protonated peptides observed upon supplemental IR activation of the chemical noise decreased with higher inlet capillary temperatures. Collision activation was not as effective for desolvating the ions present in the chemical noise.