Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Lipid/peptide/nucleotide separation with MALDI-ion mobility-TOF MS

Matrix-assisted laser desorption/ionization when combined with ion mobility-orthogonal time-of-flight mass spectrometry is a viable technique for fast separation and analysis of biomolecules in complex mixtures. Isobaric lipid, peptide, and oligonucleotide ions are preseparated before mass analysis by differences of up to 30% in mobility drift time. Ions of similar chemical type fall along well-defined "trend lines" (with deviations of approximately 3%) when plotted in two-dimensional representations of ion mobility as a function of m/z. Discussion of fundamental and technical limitations of the technique point to its potential for being most useful when applied to systems such as bodily fluids and intact tissue, where an alternative chemical or chromatographic preseparation step prior to mass analysis is either impractical or undesirable.     

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.     

Rapid separation and quantitative analysis of peptides using a new nanoelectrospray- differential mobility spectrometer-mass spectrometer system

Differential mobility spectrometry (DMS) (see Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148), also commonly referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS) (see Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszcyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105), is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal-to-noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we investigate the use of our nanoESI-DMS-MS system to demonstrate differential mobility separation of peptides. The formation of higher order peptide aggregate ions (ion complexes) via electrospray ionization and the negative impact this has on DMS peptide separation are examined. The successful use of differential mobility drift gas modifiers (dopants) to reduce aggregate ion size and improve DMS peptide ion separation is presented. Following optimization of DMS peptide separation conditions, we examined next the feasibility of a new analytical platform which uses direct sample infusion with nanoESI-DMS-MS for ultrarapid analyte quantitation. Quantitation of a selected peptide from a semicomplex peptide mixture is presented. Initial feasibility results with this new approach demonstrate good accuracy and reproducibility, as well as an absolute mass sensitivity of 6.8 amol and a minimum dynamic range of 2500 for the peptide of interest. This report offers a first look at utilizing nanoESI-DMS-MS to create an ultrarapid (under 5 s) quantitative analysis platform and its potential in the high-throughput arena. Each ion separation technique, DMS and MS, offers orthogonal ion separation to one another, enhancing the overall specificity for this quantitative approach.     

Surface-induced dissociation of ion mobility-separated noncovalent complexes in a quadrupole/time-of-flight mass spectrometer

A custom in-line surface-induced dissociation (SID) device has been incorporated into a commercial ion mobility quadrupole/time-of-flight mass spectrometer in order to provide an alternative and potentially more informative activation method than the commonly used collision-induced dissociation (CID). Complicated sample mixtures can be fractionated by ion mobility (IM) and then dissociated by CID or SID for further structural analysis. Interpretation of SID spectra for cesium iodide clusters was greatly simplified with IM prior to dissociation because products originating from different precursors and overlapping in m/z but separated in drift time can be examined individually. Multiple conformations of two protein complexes, source-activated transthyretin tetramer and nativelike serum amyloid P decamer, were separated in ion mobility and subjected to CID and SID. CID spectra of the mobility separated conformations are similar. However, drastic differences can be observed for SID spectra of different conformations, implying different structures in the gas phase. This work highlights the potential of utilizing IM-SID to study quaternary structures of protein complexes and provides information that is complementary to our recently reported SID-IM approach.     

Gas-phase separations of electrosprayed peptide libraries.

High-resolution ion mobility spectrometry has been combined with time-of-flight mass spectrometry for analysis of a combinatorial peptide library that is expected to contain 676 components. In this approach, the components of a mixture of three residue peptides, having the general form (D)Phe-Xxx-Xxx-CONH2 (where Xxx is randomized over 26 residues including 10 naturally occurring amino acids and 16 synthetic forms) were ionized by electrospray ionization. Ion mobility/time-of-flight distributions have been recorded for all ions using a nested drift(flight) time technique. The improvement in resolving power [(t/delta t) = 100-150 for singly charged ions] was illustrated by analysis of a mixture of tryptic digest peptides using high- and low-resolution instruments. The approach allows many components of the library (e.g., structural, sequence, and stereo isomers) that cannot be distinguished by mass spectrometry alone to be resolved. Impurities due to side reactions appear to be minimal, comprising < 10% of the total ion signal. Direct evidence for approximately 60-70% of the expected peptides is found. Variation in ion abundance for different components indicates that there are differences in solution concentrations or ionization efficiencies for the components.     

CIFTER: automated charge-state determination for peptide tandem mass spectra

Tandem mass spectrometry (MS/MS) has become a common and useful tool for analyzing complex protein mixtures. Database search programs are the most popular means for peptide identification from MS/MS spectra. However, estimations of charge states of peptide MS/MS spectra obtained from low-resolution mass spectrometers have not been reliable. They require repetitive database searches and additional analyses of the search results. We propose here an algorithm designed to reliably differentiate doubly charged spectra from triply charged ones. We conducted a rigorous analysis of various spectral features and their effects. We employed the distinguishing features found in our analysis and developed a classifier for multiply charged spectra using a machine learning approach. The test on various data sets showed that our method could be successfully applied independent of experimental setup and mass instrument. This algorithm can be used to prefilter spectra so that only reasonably good spectra are submitted to database search programs, thereby saving considerable time. The software for MS/MS charge-state determination, which we named "CIFTER", is available at a website http://prix.uos.ac.kr/sifter/cifter.     

An IMS-IMS analogue of MS-MS

The development of a new ion mobility/mass spectrometry instrument that incorporates a multifield drift tube/ion funnel design is described. In this instrument, individual components from a mixture of ions can be resolved and selected on the basis of mobility differences prior to collisional activation inside the drift tube. The fragment ions that are produced can be dispersed again in a second ion mobility spectrometry (IMS) region prior to additional collisional activation and MS analysis. The result is an IMS-IMS analogue of MS-MS. Here, we describe the preliminary instrumental design and experimental approach. We illustrate the approach by examining the highly characterized bradykinin and ubiquitin systems. Mobility-resolved fragment ions of bradykinin show that b-type ions are readily discernible fragments, because they exist as two easily resolvable structural types. Current limitations and future directions are briefly discussed.     

IMS-IMS and IMS-IMS-IMS/MS for separating peptide and protein fragment ions

Multidimensional ion mobility spectrometry (IMS-IMS and IMS-IMS-IMS) techniques have been combined with mass spectrometry (MS) and investigated as a means of generating and separating peptide and protein fragment ions. When fragments are generated inside a drift tube and then dispersed by IMS prior to MS analysis, it is possible to observe many features that are not apparent from MS analysis alone. The approach is demonstrated by examining fragmentation patterns arising from electrospray ion distributions of insulin chain B and ubiquitin. The multidimensional IMS approach makes it possible to select individual components for collisional activation and to disperse fragments based on differences in mobility prior to MS analysis. Such an approach makes it possible to observe many features not apparent by MS analysis alone.     

Assessing the peak capacity of IMS-IMS separations of tryptic peptide ions in He at 300 K

Two-dimensional ion mobility spectrometry (IMS-IMS) coupled with mass spectrometry is examined as a means of separating mixtures of tryptic peptides (from myoglobin and hemoglobin). In this study, we utilize two distinct drift regions that are identical in that each contains He buffer gas at 300 K. The two-dimensional advantage is realized by changing the structures of the ions. As ions arrive at the end of the first drift region, those of a specified mobility are selected, exposed to energizing collisions, and then introduced into a second drift region. Upon collisional activation, some ions undergo structural transitions, leading to substantial changes in their mobilities; others undergo only slight (or no) mobility changes. Examination of peak positions and shapes for peptides that are separated in the first IMS dimension indicates experimental peak capacities ranging from approximately 60 to 80; the peak shapes and range of changes in mobility that are observed in the second drift region (after activation) indicate a capacity enhancement ranging from a factor of approximately 7 to 17. Thus, experimental (and theoretical) evaluation of the peak capacity of IMS-IMS operated in this fashion indicates that capacities of approximately 480 to 1360 are accessible for peptides. Molecular modeling techniques are used to simulate the range of structural changes that would be expected for tryptic peptide ions and are consistent with the experimental shifts that are observed.     

Performance of a linear ion trap-Orbitrap hybrid for peptide analysis

Proteomic analysis of digested complex protein mixtures has become a useful strategy to identify proteins involved in biological processes. We have evaluated the use of a new mass spectrometer that combines a linear ion trap and an Orbitrap to create a hybrid tandem mass spectrometer. A digested submandibular/sublingual saliva sample was used for the analysis. We find the instrument is capable of mass resolution in excess of 40,000 and mass measurement accuracies of less than 2 ppm for the analysis of complex peptide mixtures. Such high mass accuracy allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion of the sample should not automatically be considered as false positives. Tandem mass spectra from the linear ion trap and from the Orbitrap have very similar ion abundance ratios. We conclude this instrument will be well suited for shotgun proteomic types of analyses.     

Ion mobility mass spectrometry analysis of human glycourinome

Complex carbohydrates are macromolecules biosynthesized in nontemplate-type processes, bearing specific glycoepitopes involved in crucial recognition processes such as cell differentiation and cell-cell interactions. Chemical structure of single components in complex mixtures can be analyzed by mass spectrometry for determination of the size and sequence of monosaccharides involved, branching patterns, and substitution by fucose and sialic acids. For de novo identification of glycoforms in human urinome containing N- and O-free and amino acid-linked oligosaccharides, a novel method of ion mobility tandem mass spectrometry followed by computer-assisted assignment is described. Distinct patterns of ions nested specifically by their m/z values and their drift time are observed by IMS-MS. An additional peak capacity for identification of time-separated m/z values in the IMS TOF MS mode for differentiation of singly, doubly, and triply charged molecular ion species by ion mobility separation contributes to significant reduction of carbohydrate complexity in a given mass window. Profiling of glycoforms from human urinome represents a highly efficient approach for biomarker discovery and differential glycotarget identification, demonstrating potential for diagnosis of human diseases, as for congenital disorders of glycosylation.     

Characterization of strategies for obtaining confident identifications in bottom-up proteomics measurements using hybrid FTMS instruments

Hybrid FTMS instruments, such as the LTQ-FT and LTQ-Orbitrap, are capable of generating high duty cycle linear ion trap MS/MS data along with high resolution information without compromising the overall throughput of measurements. Combined with online LC separations, these instruments provide powerful capabilities for proteomics research. In the present work, we explore three alternative strategies for high throughput proteomics measurements using hybrid FTMS instruments. Our accurate mass and time tag (AMT tag) strategy enables identification of thousands of peptides in a single LC-FTMS analysis by comparing accurate molecular mass and LC elution time information from the analysis to a reference database. An alternative strategy considered here, termed accurate precursor mass filter (APMF), employs linear ion trap (low resolution) MS/MS identifications generated by an appropriate search engine, such as SEQUEST, refined with high resolution precursor ion data obtained from FTMS mass spectra. The APMF results can be additionally filtered using the LC elution time information from the AMT tag database, which constitutes a precursor mass and time filter (PMTF), the third approach implemented in this study. Both the APMF and the PMTF approaches are evaluated for coverage and confidence of peptide identifications and contrasted with the AMT tag strategy. The commonly used decoy database method and an alternative method based on mass accuracy histograms were used to reliably quantify identification confidence, revealing that both methods yielded similar results. Comparison of the AMT, APMF and PMTF approaches indicates that the AMT tag approach is preferential for studies desiring a highest achievable number of identified peptides. In contrast, the APMF approach does not require an AMT tag database and provides a moderate level of peptide coverage combined with acceptable confidence values of approximately 99%. The PMTF approach yielded a significantly better peptide identification confidence, >99.9%, that essentially excluded any false peptide identifications. Since AMT tag databases that exclude incorrect identifications are desirable, this study points to the value of a multipass APMF approach to generate AMT tag databases, which are then validated using the PMTF approach. The resulting compact, high quality databases can then be used for subsequent high-throughput, high peptide coverage AMT tag studies.     

Guanidino labeling derivatization strategy for global characterization of peptide mixtures by liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

Guanidination performed with isotopic isoforms of O-methylisourea was used in combination with reversed-phase liquid chromatography (LC) matrix-assisted laser desorption/ionization to characterize, both qualitatively and quantitatively, protein mixtures. Synthesis of (13)C- and (15)N(2)-labeled O-methylisourea sulfate produces a molecule that is 3 Da heavier than the light isotopic variant. Protein mixtures containing identical components in different concentration are pooled together following parallel derivatization. Relative quantification of protein mixtures is achieved by mass spectrometry. A difference of 3 Da allows negligible interference between the two isotopic clusters for quantification of peptides up to 1400 Da. Under these conditions, the chromatographic resolution achieved allows separation of different pairs of derivatized peptides without altering the retention time of structurally identical isotopic isoforms. Concomitant isolation of both chemically modified precursors is followed by tandem mass analysis. Activation of the ions via collisions with an inert gas produces isotopically derivatized fragment ions, which appear as doublets in the product ion spectrum. Since the modification occurs on the C-terminal lysine, ions incorporating the guanidino moiety on the C-terminus can be distinguished from those containing the original unmodified peptide N-terminus. Knowledge of the location of the proton can be beneficial to data interpretation and peptide sequencing.     

Qualitative and semiquantitative analysis of composite mixtures of antibodies by native mass spectrometry

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.     

Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry

In this paper, the first examples of baseline separation of isomeric macromolecules by electrospray ionization/ion mobility spectrometry (ESI/IMS) at atmospheric pressure are presented. The behavior of a number of different isomeric peptides in the IMS was investigated using nitrogen as a drift gas. The IMS was coupled to a quadrupole mass spectrometer, which was used for identification and selective detection of the electrosprayed ions. The mobility data were used to determine their average collision cross sections. The gas-phase ions of isomeric peptides were found to have different collision cross sections. In all cases, doubly charged ions exhibited significantly (8-20%) larger collision cross sections than the respective singly charged species. The analysis of mixtures of the isomeric peptides clearly demonstrated the capability of IMS to separate gas-phase peptide ions due to small differences in their conformational structures, which cannot be determined by mass spectrometry. An actual resolving power of 80 was achieved for two doubly charged reversed sequenced pentapeptides. Baseline separation was provided for ions differing by only 2.5% in their measured collision cross sections; partial separation was shown for isomeric ions exhibiting differences as small as 1.1%.     

A correlation algorithm for the automated quantitative analysis of shotgun proteomics data

Quantitative shotgun proteomic analyses are facilitated using chemical tags such as ICAT and metabolic labeling strategies with stable isotopes. The rapid high-throughput production of quantitative "shotgun" proteomic data necessitates the development of software to automatically convert mass spectrometry-derived data of peptides into relative protein abundances. We describe a computer program called RelEx, which uses a least-squares regression for the calculation of the peptide ion current ratios from the mass spectrometry-derived ion chromatograms. RelEx is tolerant of poor signal-to-noise data and can automatically discard nonusable chromatograms and outlier ratios. We apply a simple correction for systematic errors that improves the accuracy of the quantitative measurement by 32 +/- 4%. Our automated approach was validated using labeled mixtures composed of known molar ratios and demonstrated in a real sample by measuring the effect of osmotic stress on protein expression in Saccharomyces cerevisiae.     

Electrospray ionization high-resolution ion mobility spectrometry for the detection of organic compounds, 1. Amino acids

Our aim in this investigation was to demonstrate the potential of the high-resolution electrospray ionization ion mobility spectrometry (ESI-IMS) technique as an analytical separation tool in analyzing biomolecular mixtures to pursue astrobiological objectives of searching for the chemical signatures of life during an in-situ exploration of solar system bodies. Because amino acids represent the basic building blocks of life, we used common amino acids to conduct the first part of our investigation, which is being reported here, to demonstrate the feasibility of using the ESI-IMS technique for detection of the chemical signatures of life. The ion mobilities of common amino acids were determined by electrospray ionization ion mobility spectrometry using three different drift gases (N2, Ar, and CO2). We demonstrated that the selectivity can be vastly improved in ion mobility spectroscopy (IMS) in detecting organic molecules by using different drift gases. When a judicial choice of drift gas is made, a vastly improved separation of two different amino acid ions resulted. It was found that each of the studied amino acids could be uniquely identified from the others, with the exception of alanine and glycine, which were never separable by more then 0.1 ms. This unique identification is a result of the different polarizabilities of the various drift gases. In addition, a better separation was achieved by changing the drift voltage in successive experimental runs without significantly degrading the resolution. We also report the result of our analysis of liquid samples containing mixtures of amino acids.     

Screening method for isopeptides from small ubiquitin-related modifier-conjugated proteins by ion mobility mass spectrometry

Posttranslational modification by the small ubiquitin-related modifier (SUMO) is a highly regulated modification, which is often restricted to very specific cellular events. A number of analytical strategies for identification of SUMOylated proteins have been previously reported in the literature. A new screening method for SUMOylated peptides based on ion mobility mass spectrometry is presented. Using poly-SUMO2 as a model system, a two-enzyme trypsin/chymotrypsin digestion was performed to reduce the size of the isopeptide conjugated to the substrate lysine residue. Traveling wave ion mobility mass spectrometry was used to screen for peptides containing the QQQTGG isopeptide tag from SUMO, which increases the mass and size of the peptide by 618 Da. This increase in mass along with solution conditions to promote higher charge states allows the isopeptides to be separated from the typically smaller and lesser charged linear peptides. On the basis of these findings, this method can be used as a quick and easy screening method for identifying possible SUMO isopeptides.