Multiply Charged Peptides Research Articles

Ultrahigh-resolution differential ion mobility spectrometry using extended separation times.

Ion mobility spectrometry (IMS), and particularly differential IMS or field asymmetric waveform IMS (FAIMS), is emerging as a versatile tool for separation and identification of gas-phase ions, especially in conjunction with mass spectrometry. For over two decades since its inception, the utility of FAIMS was constrained by resolving power (R) of less than ∼20. Stronger electric fields and optimized gas mixtures have recently raised achievable R to ∼200, but further progress with such approaches is impeded by electrical breakdown. However, the resolving power of planar FAIMS devices using any gas and field intensity scales as the square root of separation time (t). Here, we extended t from the previous maximum of 0.2 s up to 4-fold by reducing the carrier gas flow and increased the resolving power by up to 2-fold as predicted, to >300 for multiply charged peptides. The resulting resolution gain has enabled separation of previously "co-eluting" peptide isomers, including folding conformers and localization variants of modified peptides. More broadly, a peak capacity of ∼200 has been reached in tryptic digest separations.

Atmospheric pressure‐electron capture dissociation of peptides using a modified PhotoSpray ion source

An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévote's group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.

Evaluation of Phenylthiocarbamoyl-Derivatized Peptides by Electrospray Ionization Mass Spectrometry: Selective Isolation and Analysis of Modified Multiply Charged Peptides for Liquid Chromatography−Tandem Mass Spectrometry Experiments

Edman degradation in the gas phase has been observed by collision activated dissociation of N-terminal phenylthiocarbamoyl (PTC) protonated peptide to yield abundant complementary b₁ and y(n-1) ion pairs. Here, we demonstrated the relation between the observed losses of aniline and/or the entire PTC derivatizing group with the availability of mobile protons using electrospray ionization mass spectrometry. In order to select the peptides with more efficient fragmentation, while simplifying the mixture of peptides, we extend the phenylisotiocyanate (PITC) derivatization of amino groups to the selective isolation of multiply charged peptides (those having the number of arginines and histidines residues higher than one) using a procedure previously developed in our group. Thus, it was possible to identify in the filtered protein database the sequence of the isolated multiply charged peptides derived from a single protein and a complex mixture of proteins extracted from Escherichia coli using only the molecular mass and the N-terminal amino acid information. For this purpose, we developed a novel bioinformatic tool for automatic identification of peptides from liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, which potentially can be used in high-throughput proteomics.

Dimethyl Isotope Labeling Assisted De Novo Peptide Sequencing

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.

Influence of Charge State and Amino Acid Composition on Hydrogen Transfer in Electron Capture Dissociation of Peptides

Although conventional N-Cα bond cleavage in electron capture dissociation (ECD) of multiply-charged peptides generates a complementary c' and z(·) fragment pair, the N-Cα cleavage followed by hydrogen transfer from c' to z(·) fragments produces other fragments, namely c(·) and z'. In this study, the influence of charge state and amino acid composition on hydrogen transfer in ECD is described using sets of peptides. Hydrogen transferred ionic species such as c(·) and z' were observed in ECD spectra of doubly-protonated peptides, while the triply-protonated form did not demonstrate hydrogen transfer. The extent of hydrogen transfer in ECD of doubly-protonated peptides was dependent on constituent amino acids. The ECD of doubly-protonated peptides possessing numerous basic sites showed extensive hydrogen transfer compared with ECD of less basic peptides. The extent of hydrogen transfer is discussed from the viewpoints of the structure of peptide ions, the possibility of internal hydrogen bonding and intermediate lifetime of complex [c' + z(·)].

High-resolution differential ion mobility separations using planar analyzers at elevated dispersion fields.

The ion mobility spectrometry (IMS) methods are grouped into conventional IMS, based on the absolute ion mobility, and differential or field asymmetric waveform IMS (FAIMS), based on mobility differences between strong and weak electric fields. A key attraction of FAIMS is substantial orthogonality to mass spectrometry (MS). Although several FAIMS/MS platforms were commercialized, their utility was limited by FAIMS resolving power, typically ∼10-20. Recently, gas mixtures comprising up to 75% He have enabled resolving power >100 that permits separation of numerous heretofore "coeluting" isomers. This performance opens major new proteomic and other biological applications. Here, we show that raising the separation field by ∼35% over the previous 21 kV/cm provides similar or better resolution (with resolving powers of >200 for multiply charged peptides) using only 50% He, which avoids problems due to elevated pressure and He content in the mass spectrometer. The heating of ions by the separation field in this regime exceeds that at higher He content but weaker field, inducing greater isomerization of labile species.

Ultrasonication-Assisted Spray Ionization Mass Spectrometry for the Analysis of Biomolecules in Solution

In this paper, we describe a novel technique--ultrasonication-assisted spray ionization (UASI)--for the generation of singly charged and multiply charged gas-phase ions of biomolecules (e.g., amino acids, peptides, and proteins) from solution; this method employs a low-frequency ultrasonicator (ca. 40 kHz) in place of the high electric field required for electrospray ionization. When a capillary inlet is immersed into a sample solution within a vial subjected to ultrasonication, the solution is continually directed to the capillary outlet as a result of ultrasonication-assisted capillary action; an ultrasonic spray of the sample solution is emitted at the outlet of the tapered capillary, leading to the ready generation of gas-phase ions. Using an ion trap mass spectrometer, we found that singly charged amino acid and multiply charged peptides/proteins ions were generated through this single-step operation, which is both straightforward and extremely simple to perform. The setup is uncomplicated: only a low-frequency ultrasonicator and a tapered capillary are required to perform UASI. The mass spectra of the multiply charged peptides and proteins obtained from sample solutions subjected to UASI resemble those observed in ESI mass spectra.

Selective Isolation—Detection of Two Different Positively Charged Peptides Groups by Strong Cation Exchange Chromatography and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Application to Proteomics Studies

We report here a procedure for the independent analysis of two groups of peptides by liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS/MS), using a selective isolation-detection procedure. In this procedure all primary amino groups of tryptic peptides derived from mouse liver proteins are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply charged peptides (R + H > 1) are retained on the column and separated with high selectivity from singly (R + H = 1) and neutral peptides (R + H = 0) which are together collected in the flow-through. Using LC-MALDI-MS/MS analysis, the retained fraction displayed a 94% of enrichment of multiply charged peptides while in the flow-through; peptides with at least one arginine or histidine residue were exclusively identified, which suggests that MS detection in this fraction is restricted only to those peptides with ionizable side chains, arginine and histidine amino acids.

Simplifying Fragmentation Patterns of Multiply Charged Peptides by N-Terminal Derivatization and Electron Transfer Collision Activated Dissociation

N-terminal peptide derivatization strategies used in conjunction with tandem mass spectrometry to yield simplified fragmentation patterns have shown limited success for the de novo sequencing of multiply charged peptides, including those predominantly formed in LC-ESI-MS experiments. Significant proton mobilization occurs for multiply charged peptides upon collisional activation, resulting in the formation of both N-terminal and C-terminal product ions rather than an exclusive series of C-terminal ions preferred for de novo sequencing algorithms. To circumvent this problem, multiply charged, N-terminally derivatized peptides were subjected to electron transfer reactions with fluoranthene anions to produce singly charged, radical species. Upon subsequent "soft" collision induced dissociation (CID), highly abundant z-type ions were formed nearly exclusively, which yielded simplified fragmentation patterns amenable to de novo sequencing methods. Furthermore, the derivatized peptides retained labile phosphoric acid moieties, and the enhanced set of z ions were also observed for peptides not possessing basic C-terminal residues, a type of peptide that poses more challenges to traditional simplification methods based on collision activated dissociation. This improved LC-MS(n) strategy was demonstrated for a variety of multiply charged model peptides and a tryptic digest of myoglobin.

Generation of Multiply Charged Peptides and Proteins by Radio Frequency Acoustic Desorption and Ionization for Mass Spectrometric Detection

The design and implementation of a radio frequency acoustic desorption ionization (RADIO) source has been demonstrated for the analysis of multiply charged peptides and proteins. One muL aliquots of melittin, BNP-32, and ubiquitin ( approximately 1 mug of analyte) were deposited onto a quartz crystal microbalance (QCM) electrode before radio frequency actuation for desorption. Continuous electrospray parallel to/above the sampling surface enabled the ionization of desorbed species. Detection by a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer confirmed the intact and dissociated species observed during MS and MS/MS experiments, respectively.

Exploring Deprotonation Reactions on Peptides and Proteins at Atmospheric Pressure by Electro-Sonic Spray Ionization-Mass Spectrometry (ESSI-MS)

Electrospray ionization mass spectrometry (ESI-MS) rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For peptides and proteins, ESI leads to the formation of multiply charged ions, in both the positive and negative ion mode. The charge-state distribution is directly related to the conformation of the macromolecular ions and to proton transfer reactions in the gas phase. Deprotonation reactions of multiply charged peptide and protein ions were studied by introducing volatile reference bases at atmospheric pressure between an electro-sonic spray ionization (ESSI) source and the inlet of a mass spectrometer. This new set-up offers the unique possibility to measure the apparent gas-phase basicity GBapp of multiply charged ions by a bracketing approach. The proof of principle was made using bradykinin derivatives, substance P and insulin chain B. We obtained values in excellent agreement with known GBapp values obtained at low pressure. These experiments were extended to seven model proteins showing that the thermodynamical properties of protein ions are directly correlated to the amino-acid sequence and the conformation in the gas phase. We also demonstrated that salt bridges between ionized basic and acidic sites still exist in the gas phase, confirming that electro-sonic spray is a very soft ionization technique.

Analysis of proteins and peptides on a chromatographic timescale by electron‐transfer dissociation MS

Peptide and protein sequence analysis using a combination of gas-phase ion-ion chemistry and tandem MS is described. Samples are converted to multiply charged ions by ESI and then allowed to react with fluoranthene radical anions in a quadrupole linear ion trap mass spectrometer. Electron transfer from the radical anion to the multiply charged peptide or protein promotes random fragmentation along the amide backbone that is independent of peptide or protein size, sequence, or the presence of post-translational modifications. Examples are provided that demonstrate the utility of electron-transfer dissociation for characterizing post-translational modifications and for identifying proteins in mixtures on a chromatographic timescale (500 ms/protein).

Why b, y's? Sodiation-induced tryptic peptide-like fragmentation of non-tryptic peptides

Tryptic peptides are the analytes of choice for mass spectrometric analysis of protein and peptide as they display a favorable fragmentation pattern due to the presence of a C-terminal basic amino acid residue. Clean y fragment ion series is most commonly observed for these species. In contrast, non-tryptic peptides with undefined locations of basic amino acid residues give rise to a mixture of b and y fragment ions, often preventing unambiguous assignment of fragment ion types, which in turn impedes the interpretation of the product ion spectra. Here we report that the fragmentation pattern of multiply charged non-tryptic peptides can be modulated by fragmenting the monosodiated multiply charged species instead of the multiply protonated species. Even when b fragment ions dominate the product ion spectrum of the protonated species due to the presence of a charge sequestering basic residue at the N-terminus, mainly singly charged sodium cationized y fragment ions [y n + Na] + are observed upon fragmentation of the cationized species, i.e., tryptic peptide-like fragmentation of non-tryptic peptides is achieved. Several examples of this fragmentation pattern are described, thus strongly suggesting that sodium cation may be complexed near or at the C-terminus even in the presence of other acidic residues within the peptide. This effect is especially pronounced in the case of the doubly charged non-tryptic peptides. This controlled modulation of the fragmentation behavior of non-tryptic peptides is shown to be advantageous for the de novo sequencing of non-tryptic bioactive peptides as it facilitates the differentiation between b and y ions.

Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers [1, 2], the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis.

Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomics

A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R+H<or=1) that are neither retained nor analyzed, whereas the multiply charged peptides (R+H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.

De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal Dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.

Quantitative Proteomic Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in Transcript and Protein Levels in Transformed Cells

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

SO 2−· Electron Transfer Ion/Ion Reactions with Disulfide Linked Polypeptide Ions

Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2-* in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)-S* and Chain A (or B)-SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+* ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+* ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+* ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.

Quantification of sifuvirtide in monkey plasma by an on‐line solid‐phase extraction procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry

A simple, automated and rapid method has been developed for the determination of a novel antiviral peptide sifuvirtide in monkey plasma. Raw plasma samples were directly loaded onto an on-line solid-phase extraction (SPE) column, which removes the time-consuming and laborious sample pretreatment. Following a timed valve-switching event, the analyte was eluted on-line to a reversed-phase high-performance liquid chromatography (RP-HPLC) column and subsequently introduced into a linear ion trap mass spectrometer, LTQ-MS, via an electrospray ionization (ESI) interface. The multiply charged peptides were specified and quantitatively analyzed using selective reaction monitoring (SRM). A highly pure four iodine-sifuvirtide was synthesized using an optimized iodogen method and proved to be a suitable internal standard (IS). A single analysis run takes about 18 min. Validation of the method demonstrated that the linear calibration curves covered the range of 4.88-5000 ng/mL, and the correlation coefficients were above 0.9923. The limit of detection (LOD) with the signal-to-noise (S/N) ratio higher than 12 was calculated as 1.22 ng/mL. The intra- and inter-batch precisions were less than 12.7% and 9.1%, and the mean accuracy ranged from -5.2% to 3.6%, respectively. Any carry-over effect from the system was negligible. In a pharmacokinetic (PK) study of sifuvirtide after a single intravenous or subcutaneous dose in monkeys, the on-line SPE-LC/MS/MS system was successfully utilized to determine hundreds of samples with only one extraction column, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of peptide drugs.

Electron Transfer Dissociation of Peptide Anions

Ion/ion reactions of multiply deprotonated peptide anions with xenon radical cations result in electron abstraction to generate charge-reduced peptide anions containing a free-radical site. Peptide backbone cleavage then occurs by hydrogen radical abstraction from a backbone amide N to facilitate cleavage of the adjacent C-C bond, thereby producing a- and x-type product ions. Introduction of free-radical sites to multiply charged peptides allows access to new fragmentation pathways that are otherwise too costly (e.g., lowers activation energies). Further, ion/ion chemistry, namely electron transfer reactions, presents a rapid and efficient means of generating odd-electron multiply charged peptides; these reactions can be used for studying gas-phase chemistries and for peptide sequence analysis.