LC-MS accurate quantification of a tryptic peptide co-eluted with an isobaric interference by using in-source collisional purification.

Interferences from isobaric and isomeric compounds represent a common problem in liquid chromatography coupled to mass spectrometry (LC-MS). In this paper, in-source purification and chromatographic separation were combined with the aim of identifying isobaric contamination and quantifying accurately a compound despite the presence of an isobaric co-eluted interference. This is achieved by totally fragmenting in-source the precursor ions of the isobaric interference providing then LC-pseudo-MS2 capability, which allows an accurate quantification without the need for optimizing the chromatographic conditions to separate the co-eluted interference. To illustrate this concept, mixtures of tryptic and non-tryptic peptides were used. The ratio of peak areas of the tryptic peptide and its isotopically labelled internal standard was used not only for quantification with an internal standard calibration curve but also to know (1) if an isobaric interference co-eluted with the tryptic peptide; and (2) what is the minimum cone voltage necessary to ensure the complete removal of isobaric interference. This strategy was applied to quantify the tryptic peptide of two standards with known concentrations and, intentionally contaminated with the isobaric interference. The confidence intervals of the concentrations calculated with the internal standard calibration curve were 8.0 ± 0.5 µM (prepared at 8.0 µM) and 15.7 ± 0.5 µM (prepared at 16.1 µM) that confirm the tryptic peptide can be correctly quantified by in-source purification without the need for improving the chromatographic separation from its isobaric interference.

Revealing C-terminal peptide amidation by the use of the survival yield technique.

α-amidation of peptide sequences is a common post-translational modification in the living world. Since the majority of these C-terminal amidated peptides are bioactive, there is hence a great interest to identify and characterize them from biological matrices and natural extracts. Regarding conventional separative methods dedicated to peptides (such as HPLC or CE), elution protocols must be carefully optimized hampering straightforward LC-MS analysis of complex samples. From a mass spectrometry point of view, they are difficult to pinpoint owing to the only 1 Da mass difference between the post-translational amidated and the corresponding native carboxylated forms producing overlapping isotopic contributions of both molecular ions. To circumvent this analytical difficulty, usage of energy-resolved tandem mass spectrometry experiments and of the survival yield technique was investigated. Pair of peptides were thus dissociated in positive and negative mode according to the survival yield technique, in MS2 and MS3 experiments, in order to separate them giving a reliable MS/MS methodology to detect such post-translationally modified sequence.

Phosphonodithioester-amine coupling in water: a fast reaction to modify the surface of liposomes.

The incorporation of lipophilic phosphonodithioesters in phospholipid formulations generates clickable liposomes that react with amines. The kinetics of this metal free phosphonodithioester-amine coupling (PAC) on liposomes in water is reported and can be classified as a fast reaction with a second order rate constant of k ≈ 8 × 102 M-1 s-1. The PAC reaction represents a versatile strategy to functionalize liposomes.

Exploring Phosphine Electronic Effects on Molybdenum Complexes: A Combined Photoelectron Spectroscopy and Energy Decomposition Analysis Study.

In organometallic chemistry, especially in the catalysis area, accessing the finest tuning of a catalytic reaction pathway requires a detailed knowledge of the steric and electronic influences of the ligands bound to the metal center. Usually, the M-L bond between a ligand and metal is depicted by the Dewar-Chatt-Duncanson model involving two opposite interactions, σ-donor and π-acceptor effects of the ligand. The experimental evaluation of these effects is essential and complementary to in-depth theoretical approaches that are able to provide a detailed description of the M-L bond. In this work, we present a study of LMo(CO)5 complexes with L being various tertiary phosphine ligands by means of mass-selected high-resolution photoelectron spectroscopy (PES) performed with synchrotron radiation, DFT, and energy decomposition analyses (EDA) combined with the natural orbitals for chemical valence (NOCV) analysis. These methods enable a separated access of the σ-donor and π-acceptor effects of ligands by probing either the electronic configuration of the complex (PES) or the interaction of the ligand with the metal (EDA). Three series of PR3 ligands with various electronic influences are investigated: the strong donating alkyl substituents (PMe3, PEt3, and PiPr3), the intermediate PPhxMe(3-x) (x = 0-3) set, and the PPhxPyrl(3-x) set (x = 0-3 with Pyrl being the strong electron withdrawing pyrrolyl group C4H4N). For each complex, their adiabatic and vertical ionization energies (IEs) could be determined with a 0.03 eV precision. Experiment and theory show an excellent agreement, either for the IE determination or electronic effect analysis. The ability to interpret the spectra is shown to depend on the character of the ligand. "Innocent" ligands provide the spectra that are the most straightforward to analyze, whereas the "non-innocent" ligands (which are ionized prior to the metal center) render the analysis more difficult due to an increased number of molecular orbitals in the energy range considered. A very good linear correlation is finally found between the measured adiabatic ionization energies and the interaction energy term obtained by EDA for each of these two types of ligands, which opens interesting perspective for the prediction of ligand characters.

Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum.

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.

Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Quantification of intramolecular click chemistry modified synthetic peptide isomers in mixtures using tandem mass spectrometry and the survival yield technique.

Intramolecular click-chemistry is increasingly used to generate and control the architecture of complex macromolecules including peptides. Such compounds are, however, very challenging to analyze, in particular quantitatively and also to assess their purity. In this study, tandem mass spectrometry (MS/MS) experiments were carried out with an ion trap mass spectrometer using the Survival Yield (SY) technique to analyze several mixtures of protonated, alkali and alkaline earth metal complexes of two topological linear and cyclic peptide isomers. Univariate (using a single excitation voltage) and multivariate (using several excitation voltages) calibration models have been used. The sensitivity, linearity (R2), intermediate precision (sInt) and error of predicted values (RMSEP) of external calibrations curves have been compared leading to the conclusions that: 1) quantification using tandem mass spectrometry can be performed, with very good performances, for such peptides despite isomerism, 2) quantification is also possible despite the absence of diagnostic fragment ions (possibly independently of the amino-acid sequence), 3) best results are obtained with the largest alkali cation, Cs+, while protonation is highly discouraged, 4) uni/multivariate models show similar performances, but the univariate model may be more suitable for potential applications with direct infusion by electrospray ionization (ESI-MS/MS) and/or matrix-assisted laser desorption ionization (MALDI-MS/MS). Graphical abstract ᅟ.

Molecular Level Structure of Biodegradable Poly(Delta-Valerolactone) Obtained in the Presence of Boric Acid.

In this study, low molecular weight poly(δ-valerolactone) (PVL) was synthesized through bulk-ring openings polymerization of δ-valerolactone with boric acid (B(OH)3) as a catalyst and benzyl alcohol (BnOH) as an initiator. The resulting homopolymer was characterized with the aid of nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques to gain further understanding of its molecular structure. The electrospray ionization mass spectrometry (ESI-MS) spectra of poly(δ-valerolactone) showed the presence of two types of homopolyester chains-one terminated by benzyl ester and hydroxyl end groups and one with carboxyl and hydroxyl end groups. Additionally, a small amount of cyclic PVL oligomers was identified. To confirm the structure of PVL oligomers obtained, fragmentation of sodium adducts of individual polyester molecules terminated by various end groups was explored in ESI-MSn by using collision induced dissociation (CID) techniques. The ESI-MSn analyses were conducted both in positive- and negative ion mode. The comparison of the fragmentation spectra obtained with proposed respective theoretical fragmentation pathways allowed the structure of the obtained oligomers to be established at the molecular level. Additionally, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), it was proven that regardless of the degree of oligomerization, the resulting PVL samples were a mixture of two types of linear PVL oligomers differing in end groups and containing just a small amount of cyclic oligomers that tended to be not visible at higher molar masses.

Understanding the CO Dissociation in [Fe(CN)2(CO)2(dithiolate)]2- Complexes with Quantum Chemical Topology Tools.

The active site of the [NiFe]-hydrogenase contains a pentacoordinated iron atom; therefore, a vacant coordination site is available for substrate binding. Nonetheless, most organometallic models of the [NiFe]-hydrogenase failed to reproduce this key feature of the active site. In order to rationalize such behavior, quantum chemical calculations were carried out on a series of [Fe(CN)2(CO)n(dithiolate)]2- n = 1,2 complexes, where dithiolate denotes the ligands (CF3)2C2S22-, (CO2Me)2C2S22-, Ph2C2S22-, C6Cl2H2S22-, C6H4S22-, C2H4S22-, and C3H6S22-. Structural and energetic features are discussed, and a topological analysis based on two scalar fields, the one-electron density and the electron localization function (ELF), has been attempted to describe the nature of the metal-ligand bonds. The present approach contributes to better understand the ability of noninnocent dithiolene to strongly labilize one CO whereas innocent dithiolate cannot. The methodology developed throughout the paper could be useful in the field of the CO-releasing molecules.

Purification and Quantification of an Isomeric Compound in a Mixture by Collisional Excitation in Multistage Mass Spectrometry Experiments.

The differentiation, characterization, and quantification of isomers and/or isobars in mixtures is a recurrent problem in mass spectrometry and more generally in analytical chemistry. Here we present a new strategy to assess the purity of a compound that is susceptible to be contaminated with another isomeric side-product in trace levels. Providing one of the isomers is available as pure sample, this new strategy allows the detection of isomeric contamination. This is done thanks to a "gas-phase collisional purification" inside an ion trap mass spectrometer paving the way for an improved analysis of at least similar samples. This strategy consists in using collision induced dissociation (CID) multistage mass spectrometry (MS2 and MS3) experiments and the survival yield (SY) technique. It has been successfully applied to mixtures of cyclic poly(L-lactide) (PLA) with increasing amounts of its linear topological isomer. Purification in gas phase of PLA mixtures was established based on SY curves obtained in MS3 mode: all samples gave rise to the same SY curve corresponding then to the pure cyclic component. This new strategy was sensitive enough to detect traces of linear PLA (<3%) in a sample of cyclic PLA that was supposedly pure according to other characterization techniques (1H NMR, MALDI-HRMS, and size-exclusion chromatography). Moreover, in this case, the presence of linear isomer was undetectable according to MS/MS or MS/MS/MS analysis only as fragment ions are also of the same m/z values. This type of approach could easily be implemented in hyphenated mass spectrometric techniques to improve the structural and quantitative analysis of complex samples.

CuAAC Click Reactions in the Gas Phase: Unveiling the Reactivity of Bis-Copper Intermediates.

Copper-catalysed azide alkyne cycloaddition (CuAAC) has been considered a breakthrough transformation over the last 15 years. Its debated mechanism arouses continuously growing interest. By means of a mass spectrometer modified ad hoc, the entire catalytic cycle of CuAAC reaction has been investigated in the gas phase. Ion-molecule reactions were performed inside the mass spectrometer to reproduce step-by-step, at a molecular level, the complete catalytic cycle of the click reaction. We successfully challenged the reactivity of elusive mono- and bis-copper intermediates by ion-molecule reactions leading to the production of mass-characterized triazole products, paving the way for detailed energetic studies to be performed in the gas phase. The structures of the relevant species, calculated at a DFT level, helped rationalise our experimental results.

The Pauson-Khand mechanism revisited: origin of CO in the final product.

The mechanism of the Pauson-Khand reaction has been studied by mass spectrometry and it has been found, through ion-molecule reaction with (13) CO, that the carbon monoxide incorporated into the product cyclopentenone is one that has been retained within the complex. Theoretical and kinetic calculations support this finding, which provides a complementary explanation for the effect of Pauson-Khand promoters.

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 µM up to 2.5 µM. The detection limit was 0.08 µM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 µM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Leucine enkephalin--a mass spectrometry standard.

The present article reviews the mass spectrometric fragmentation processes and fragmentation energetics of leucine enkephalin, a commonly used peptide, which has been studied in detail and has often been used as a standard or reference compound to test novel instrumentation, new methodologies, or to tune instruments. The main purpose of the article is to facilitate its use as a reference material; therefore, all available mass spectrometry-related information on leucine enkephalin has been critically reviewed and summarized. The fragmentation mechanism of leucine enkephalin is typical for a small peptide; but is understood far better than that of most other compounds. Because ion ratios in the MS/MS spectra indicate the degree of excitation, leucine enkephalin is often used as a thermometer molecule in electrospray or matrix-assisted laser desorption ionization (ESI or MALDI). Other parameters described for leucine enkephalin include collisional cross-section and energy transfer; proton affinity and gas-phase basicity; radiative cooling rate; and vibrational frequencies. The lowest-energy fragmentation channel of leucine enkephalin is the MH(+) → b(4) process. All available data for this process have been re-evaluated. It was found that, although the published E(a) values were significantly different, the corresponding Gibbs free energy change showed good agreement (1.32 ± 0.07 eV) in various studies. Temperature- and energy-dependent rate constants were re-evaluated with an Arrhenius plot. The plot showed good linear correlation among all data (R(2) = 0.97), spanned over a 9 orders of magnitude range in the rate constants and yielded 1.14 eV activation energy and 10(11.0) sec(-1) pre-exponential factor. Accuracy (including random and systematic errors, with a 95% confidence interval) is ±0.05 eV and 10(±0.5) sec(-1), respectively. The activation entropy at 470 K that corresponds to this reaction is -38.1 ± 9.6 J mol(-1) K(-1). We believe that these re-evaluated values are by far the most accurate activation parameters available at present for a protonated peptide and can be considered as "consensus" values; results on other processes might be compared to this reference value.

A simple method to estimate relative stabilities of polyethers cationized by alkali metal ions.

Dissociation of doubly cationized polyethers, namely [P + 2X](2+) into [P + X](+) and X(+), where P = polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetrahydrofuran (PTHF) and X = Na, K and Cs, was studied by means of energy-dependent collision-induced dissociation tandem mass spectrometry. It was observed that the collision voltage necessary to obtain 50% fragmentation (CV(50)) determined for the doubly cationized polyethers of higher degree of polymerization varied linearly with the number of degrees of freedom (DOF) values. This observation allowed us to correlate these slopes with the corresponding relative gas-phase dissociation energies for binding of alkali ions to polyethers. The relative dissociation energies determined from the corresponding slopes were found to decrease in the order Na(+) > K(+) > Cs(+) for each polyether studied, and an order PPG ≈ PEG > PTHF can be established for each alkali metal ion.

Structure and energetics of poly(ethylene glycol) cationized by Li(+), Na(+), K(+) and Cs(+): a first-principles study.

Density functional theoretical methods, including several basis sets and two functional, were used to collect information on the structure and energetic parameters of poly(ethylene glycol) (PEG), also referred to as poly(ethylene oxide) (PEO), coordinated by alkali metal ions. The oligomer chain is found to form a spiral around the alkali cation, which grows to roughly two helical turns when the oligomer size increases to about the decamer for each alkali ion. Above this size, the additional monomer units do not build the spiral further for Li(+) and Na(+); instead, they form less organized segments outside or next to the initial spiral. The distance of the first layer of co-ordinating O atoms from the alkali cation is 1.9-2.15 Å for Li(+), 2.3-2.5 Å for Na(+), 2.75-3.2 Å for K(+) and 3.5-3.8 Å for Cs(+) complexes. The number of O atoms in the innermost shell is five, six, seven and eleven for Li(+), Na(+), K(+) and Cs(+). The collision cross sections with He increase linearly with the oligomer to a very good approximation. No sign of leaning towards the 2/3 power dependence characterizing spherical particles is observed. The binding energy of the cation to the oligomer increases up to polymerization degree of about 10, where it levels off for each alkali-metal ion, indicating that this is approximately the limit of the oligomer size that can be influenced by the alkali cation. The binding energy-degree of polymerization curves are remarkably parallel for the four cations. The limiting binding energy at large polymerization degrees is about 544 kJ mol(-1), 460 kJ mol(-1), 356 kJ mol(-1) and 314 kJ mol(-1) for Li, Na, K and Cs, respectively. The geometrical features are compared with the X-ray and neutron diffraction data on crystalline and amorphous phases of conducting polymers formed by alkali-metal salts and PEG. The implications of the observations concerning collision cross sections and binding energies to ion mobility spectroscopy and mass spectrometry are discussed.

The Resonant Excitation Process in Commercial Quadrupole Ion Traps Revisited.

The collision-induced resonant excitation process in real quadrupole ion traps is revisited theoretically and experimentally by explicitly including in the discussion the influence of higher order potential impurities. This includes mainly the dependence of the secular oscillation frequency fion on the ion's oscillation amplitude zmax. Due to frequency calibration, commercial ion traps use excitation frequencies fexc that are higher than the theoretical secular oscillation frequency fion. This may lead to switching in frequency order between fexc and fion that can allow ions to stay longer in on-resonance. It is also found that there is a most efficient but also a harshest excitation frequency, which are not identical. These phenomena are explained and described with a simple harmonic oscillator model and precise numerical calculations, using the trajectory simulation program ITSIM 5.0. Experimental MS2 have been performed with the thermometer ion leucine-enkephalin, which are then in line with expectations from the trajectory calculations. The important difference to the existing literature is that, here, overexcitation is characterized by the observed a4/b4 fragment-ion ratio, while the fragmentation efficiency was kept constant. By slightly increasing the excitation frequency one can obtain drastically different effective collisional temperatures. This knowledge gives even commercial ion traps, without instrument adjustments, the possibility of producing energetically versatile fragment ion spectra. It is also shown that the damped driven harmonic oscillator cannot be used as a simplified model of the motion during the resonant excitation process in real ion traps.

Structural analysis of a compound despite the presence of an isobaric interference by using in-source Collision Induced Dissociation and tandem mass spectrometry.

The presence of an isobaric contaminant can drastically affect MS and MS/MS patterns leading to erroneous structural and quantitative analysis, which is a real challenge in mass spectrometry. Herein, we demonstrate that MS and MS/MS structural analysis of a compound can be successfully performed despite the presence of an isobaric interference with as low as few millidaltons mass difference by using pseudo-MS3 . To this end, in-source collisional excitation (in-source CID) and the Survival Yield (SY) technique (energy-resolved collision induced dissociation MS/MS) were performed on two different source geometries: a Z-spray and an orthogonal spray (with a transfer capillary) ionization sources on two different mass spectrometers. By using soft ionization conditions, the SY curve for the mixture is a linear combination of the SY curves from the pure compounds demonstrating the presence of two components in the mixture. In the case of harsher ionization conditions, the SY curve of the mixture perfectly overlaps the SY curve from the pure analyte. This observation demonstrates the isobaric interference has been completely removed by in-source CID fragmentation, independently of the source design, leaving then the analyte precursor ions only. Therefore, by measuring the MS spectrum in harsh ionization conditions and according to SY criterium, the compound of interest can be made free from isobaric interference paving the way for, for example, unequivocal HPLC-MS as well as HPLC-MS/MS structural and quantitative analysis despite the presence of a co-eluting isobaric interference.

Size effect on fragmentation in tandem mass spectrometry.

The collision energy or collision voltage necessary to obtain 50% fragmentation (characteristic collision energy/voltage, CCE or CCV) has been systematically determined for different types of molecules [poly(ethylene glycols) (PEG), poly(tetrahydrofuran) (PTHF), and peptides] over a wide mass (degrees of freedom) range. In the case of lithium-cationized PEGs a clear linear correlation (R(2) > 0.996) has been found between CCE and precursor ion mass on various instrument types up to 4.5 kDa. A similar linear correlation was observed between CCV and the mass-to-charge ratio. For singly and multiply charged polymers studied under a variety of experimental conditions and on several instruments, all data were plotted together and showed correlation coefficient R(2) = 0.991. A prerequisite to observe such a good linear correlation is that the energy and entropy of activation in a class of polymers is likely to remain constant. When compounds of different structure are compared, the CCV will depend not only on the molecular mass but the activation energy and entropy as well. This finding has both theoretical and practical importance. From a theoretical point of view it suggests fast energy randomization up to at least 4.5 kDa so that statistical rate theories are applicable in this range. These results also suggest an easy method for instrument tuning for high-throughput structural characterization through tandem MS: after a standard compound is measured, the optimum excitation voltage is in a simple proportion with the mass of any structurally similar analyte at constant experimental conditions.