[Modification of cysteine residues for mass spectrometry-based proteomic analysis: facts and artifacts]. / Modifikatsiia ostatkov tsisteina dlia mass-spektrometricheskogo proteomnogo analiza: fakty i artefakty.

Mass spectrometric proteomic analysis at the sample preparation stage involves the artificial reduction of disulfide bonds in proteins formed between cysteine residues. Such bonds, when preserved in their native state, complicate subsequent enzymatic hydrolysis and interpretation of the research results. To prevent the re-formation of the disulfide bonds, cysteine residues are protected by special groups, most often by alkylation. In this review, we consider the methods used to modify cysteine residues during sample preparation, as well as possible artifacts of this stage. Particularly, adverse reactions of the alkylating agents with other amino acid residues are described. The most common alkylating compound used to protect cysteine residues in mass spectrometric proteomic analysis is iodoacetamide. However, an analysis of the literature in this area indicates that this reagent causes more adverse reactions than other agents used, such as chloroacetamide and acrylamide. The latter can be recommended for wider use. In the review we also discuss the features of the cysteine residue modifications and their influence on the efficiency of the search for post-translational modifications and protein products of single nucleotide substitutions.

Single Cell Proteogenomics - Immediate Prospects.

Recent technical advances in genomic technology have led to the explosive growth of transcriptome-wide studies at the level of single cells. The review describes the first steps of the single cell proteomics that has originated soon after development of transcriptomics methods. The first studies on the shotgun proteomics of single cells that used liquid chromatography/mass spectrometry have been already published. In these works, the cells were separated by the methods used in transcriptomics studies (e.g., cell sorting) and analyzed by modified mass spectrometry with tandem mass tags. The new proteogenomics approach involving integration of single cell transcriptomics and proteomics data will provide better understanding of the mechanisms of cell interactions in normal development and disease.

Identification of Single Amino Acid Substitutions in Proteogenomics.

An important aim of proteogenomics, which combines data of high throughput nucleic acid and protein analysis, is to reliably identify single amino acid substitutions representing a main type of coding genome variants. Exact knowledge of deviations from the consensus genome can be utilized in several biomedical fields, such as studies of expression of mutated proteins in cancer, deciphering heterozygosity mechanisms, identification of neoantigens in anticancer vaccine production, search for RNA editing sites at the level of the proteome, etc. Generation of this new knowledge requires processing of large data arrays from high-resolution mass spectrometry, where information on single-point protein variation is often difficult to extract. Accordingly, a significant problem in proteogenomic analysis is the presence of high levels of false positive results for variant-containing peptides in the produced results. Here we review recently suggested approaches of high quality proteomics data processing that may provide more reliable identification of single amino acid substitutions, especially contrary to residue modifications occurring in vitro and in vivo. Optimized methods for assessment of false discovery rate save instrumental and computational time spent for validation of interesting findings of amino acid polymorphism by orthogonal methods.

Probing the mechanisms of ambient ionization by laser-induced fluorescence spectroscopy.

The ionization mechanisms of several atmospheric pressure ion sources based on desorption and ionization of samples deposited on a surface were studied. Home-built desorption electrospray ionization (DESI), laserspray ionization (LSI), and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) sources were characterized using low-molecular-weight compounds, in particular fluorescent dyes. Detection of the desorbed and ionized species was performed by laser-induced fluorescence and ion cyclotron resonance mass spectrometry. The dependences of the signal intensities on various experimental parameters were studied. The data obtained reveals common features, such as formation of solvated species and clusters in the ionization processes, in all of the techniques considered.

Use of models of biomacromolecule separation in AMT database generation for shotgun proteomics.

Generation of a complex proteome database requires use of powerful analytical methods capable of following rapid changes in the proteome due to changing physiological and pathological states of the organism under study. One of the promising technologies with this regard is the use of so-called Accurate Mass and Time (AMT) tag peptide databases. Generation of an AMT database for a complex proteome requires combined efforts by many research groups and laboratories, but the chromatography data resulting from these efforts are tied to the particular experimental conditions and, in general, are not transferable from one platform to another. In this work, we consider an approach to solve this problem that is based on the generation of a universal scale for the chromatography data using a multiple-point normalization method. The method follows from the concept of linear correlation between chromatography data obtained over a wide range of separation parameters. The method is further tested for tryptic peptide mixtures with experimental data collected from mutual studies by different independent research groups using different separation protocols and mass spectrometry data processing tools.

Standardization of retention time data for AMT tag proteomics database generation.

The combination of liquid chromatography (LC) with mass spectrometry (MS) has become a mainstream proteome analysis strategy. In LC-MS, measured masses possess their "universal" scale derived from atomic mass tables. In contrast, the observed LC retention times (RT) are not tied to a conventional time scale, and depend on experimental conditions. However, RT data, being explicitly orthogonal to MS, offer relevant information for proteome characterization. We present here a strategy for peptides RT data standardization, based on the generation of a standard scale using retention prediction models, which enables sharing of identification databases in the context of multi-laboratory research.

A dynamic ion cooling technique for FTICR mass spectrometry.

A fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion-ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important.

Optimal pressure conditions for unbiased external ion accumulation in a two-dimensional radio-frequency quadrupole for Fourier transform ion cyclotron resonance mass spectrometry.

When combined with on-line separations (e.g., capillary liquid chromatography (LC)), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides a powerful tool for biological applications, and particularly proteomic studies. The sensitivity, dynamic range, and duty cycle provided by FTICR-MS have been shown to be increased by ion trapping and accumulation in a two-dimensional (2D) radio-frequency (rf)-only multipole positioned externally to an FTICR cell. However, it is important that ions be detected across the desired m/z range without a significant bias. In this work we found that pressure inside the accumulation rf-quadrupole plays an important role in obtaining "unbiased" ion accumulation. Pressure optimization was performed in both pulsed and continuous modes. It was found that unbiased accumulation in a 2D rf-only quadrupole could be achieved in the pressure range of 5 x 10(-4) to 5 x 10(-3) Torr. External ion accumulation performed at the optimal pressure resulted in an increase in both the spectrum acquisition rates and dynamic range.

Dynamically assisted gated trapping for Fourier transform ion cyclotron mass spectrometry.

An efficient approach for trapping ions and enhancing signal based on 'adiabatic amplitude reduction' for Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is described and evaluated. This method is a modification to the widely used gated trapping technique in which the trapping potential is raised adiabatically rather than instantaneously (non-adiabatically). Compared with non-adiabatic gated trapping, the final amplitudes of ion axial oscillations and energies are lower in the proposed method. All performance aspects of the FTICR spectrum (e.g., peak intensities, mass resolution, and mass accuracy) improve significantly compared to the conventional gated trapping technique.

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry.

Undesired fragmentation of electrospray generated ions in an rf multipole traps can be problematic in many applications. Of special interest here is ion dissociation in a 2-D quadrupole ion trap external to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) used in proteomic studies. In this work, we identified the experimental parameters that determine the efficiency of ion fragmentation. We have found that under the pressure conditions used in this study there is a specific combination of the radial and axial potential well depths that determines the fragmentation threshold. This combination of rf and dc fields appears to be universal for ions of different mass-to-charge ratios, molecular weights, and charge states. Such universality allows the fragmentation efficiency of the trapped ions in the course of capillary liquid chromatography (LC) separation studied to be controlled and can increase the useful duty cycle and dynamic range of a FTICR mass spectrometer equipped with an external rf only 2-D quadrupole ion trap.

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins.

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.

Initial implementation of an electrodynamic ion funnel with Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has become a widely used method to study biopolymers. The method, in combination with an electrospray ionization (ESI) source has demonstrated the highest resolution and accuracy yet achieved for characterization of biomolecules and their noncovalent complexes. The most common design for the ESI interface includes a heated capillary inlet followed by a skimmer having a small orifice to limit gas conductance between a higher pressure (1 to 5 torr) source region and the lower pressure ion guide. The ion losses in the capillary-skimmer interface are large (estimated to be more than 90%) and thus reduce achievable sensitivity. In this work, we report on the initial implementation of a newly developed electrodynamic ion funnel in a 3.5 tesla ESI-FTICR mass spectrometer. The initial results show dramatically improved ion transmission as compared to the conventional capillary-skimmer arrangement. An estimated detection limit of 30 zeptomoles (approximately 18,000 molecules) has been achieved for the analysis of the proteins with molecular weights ranging from 8 to 20 kDa.

Pressure limited sustained off-resonance irradiation for collision-activated dissociation in fourier transform mass spectrometry.

A theoretical evaluation of the sustained off-resonance irradiation of ions (SORI) in the presence of a collisional buffer gas in a Fourier transform ion cyclotron resonance mass spectrometer is presented. It is shown that there is an optimal pressure for a given set of irradiation parameters corresponding to the most effective dissociation. Theoretical predictions are compared with experimental results for the dissociation of electrosprayed biopolymer ions and with previously accepted view of the SORI process.

Electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: instrument design and initial results.

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10(6) has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.

A dual-trap design and its applications in electrospray ionization FTICR mass spectrometry.

A new arrangement consisting of two separate Fourier transform ion cyclotron resonance (FTICR) ion traps was used to develop methods for the manipulation of the ions produced by an electrospray ionization source (ESI). A first, "accumulation" trap, is generally maintained at a higher pressure than the second, high-performance "analyzer" trap. The manipulations developed and demonstrated include the following: (1) mass-selective ion transfers between the traps; (2) mass-selective step-wise accumulation of low-abundance ions of different mass-to-charge ratios transferred from the first trap to the analyzer trap; (3) simultaneous detection of ions in the analyzer trap and ion accumulation in the source trap; (4) simultaneous ion detection in the accumulation trap and ion storage in the analyzer trap; (5) sequential multiple transfers of the ions into the analyzer trap from the same ion population stored in the accumulation trap; (6) collision-induced dissociation of ions stored in the accumulation trap followed by mass-selective transfer of the product ions into the analyzer trap; (7) sequential transfer of the ions of different mass-to-charge ratios into the analyzer trap from the same ion population stored in the accumulation trap followed by the collision-induced dissociation of transferred ions in the analyzer trap. These ion manipulations benefit multistage studies and are projected to be useful in many biochemical applications of ESI-FTICR, including structural determination of biopolymers and study of noncovalent complexes.

A high performance low magnetic field internal electrospray ionization-fourier transform ion cyclotron resonance mass spectrometer.

A new in-magnetic field electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometer has been constructed and evaluated. This system is characterized by the use of multiple concentric cryopanels to achieve ultrahigh vacuum in the ion cyclotron resonance cell region, a probe-mounted internal ESI source, and a novel in-field shutter. Initial experiments demonstrate high resolution mass measurement capability at a field strength of 1 T. Mass resolution of 700,000 has been obtained for the 3+ charge state of Met-Lys-bradykinin (at m/z 440) generated by electrospray ionization. When electron impact ionization was employed, resolution in excess of 9,200,000 was achieved for nitrogen molecular ions (N 2 (+) ). Isotopic resolution for molecular ions of bovine ubiquitin (MW=8565 µ) also was achieved by using small ion populations.

Analysis and elimination of systematic errors originating from coulomb mutual interaction and image charge in Fourier transform ion cyclotron resonance precise mass difference measurements.

The effect of mutual Coulomb-mediated interactions between ions of two different mass-to-charge ratios (but equal ion cyclotron orbital radii) on their Fourier transform ion cyclotron resonance (FT/ICR) mass spectral frequency difference is derived analytically and measured experimentally. For a cylindrical ion trap, ion packets are modeled theoretically as infinitely extended lines of charge, and contributions to cyclotron frequency difference due to direct Coulomb repulsion between the lime charges as well as the forces arising from image charge induced on the trap electrodes by each line charge are calculated. A striking theoretical prediction is that the effect on ICR frequency difference of mutual Coulomb repulsion between ions in a mass doublet may be compensated by the image-charge effect. As a result, there is an optimal (calculable) ion cyclotron orbital radius at which the measured cyclotron orbital frequency difference between ions of two different mass-to-charge ratios is independent of mutual Coulomb-mediated interactions between the two components of the mass doublet! Moreover, if the two mass-doublet component ions are present in equal numbers, then the measured ion cyclotron orbital frequency difference is also independent of all Coulomb-mediated interactions between the two types of ions! Thus, the single largest systematic error in measurement of mass difference in a mass doublet by FT/ICR mass spectrometry may be virtually eliminated by appropriate control of ICR orbital radius and/or by performing measurements at various relative abundance ratios and extrapolating to equal relative abundance of the two mass-doublet components. We report experimental tests and verification of these predictions for two different mass doublets: (3)He(+)/(3)H(+) (cylindrical trap at 4.7 Tesla) and (12)C(1)H 2 (+) /(14) N(+) (cubic trap at 7.0 Tesla). From the latter measurement, we determine the mass of atomic nitrogen as m((14)N)=14.003 074 014(19) u.