Analysis of peptide MS/MS spectra from large-scale proteomics experiments using spectrum libraries

A widespread proteomics procedure for characterizing a complex mixture of proteins combines tandem mass spectrometry and database search software to yield mass spectra with identified peptide sequences. The same peptides are often detected in multiple experiments, and once they have been identified, the respective spectra can be used for future identifications. We present a method for collecting previously identified tandem mass spectra into a reference library that is used to identify new spectra. Query spectra are compared to references in the library to find the ones that are most similar. A dot product metric is used to measure the degree of similarity. With our largest library, the search of a query set finds 91% of the spectrum identifications and 93.7% of the protein identifications that could be made with a SEQUEST database search. A second experiment demonstrates that queries acquired on an LCQ ion trap mass spectrometer can be identified with a library of references acquired on an LTQ ion trap mass spectrometer. The dot product similarity score provides good separation of correct and incorrect identifications.     

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.     

Rectilinear ion trap: concepts, calculations, and analytical performance of a new mass analyzer

A mass analyzer based on a rectilinear geometry ion trap (RIT) has been built, and its performance has been characterized. Design concepts for this type of ion trap are delineated with emphasis on the effects of electrode geometry on the calculated electric field. The Mathieu stability region was mapped experimentally. The instrument can be operated using mass-selective instability scans in both the boundary and resonance ejection versions. Comparisons of performance between different versions of the device having different dimensions allowed selection of an optimized geometry with an appropriate distribution of higher-order electric fields. Comparisons made under the same conditions between the performance of a conventional cylindrical ion trap and a RIT of 4 times greater volume show an improvement of 40 times in the signal-to-noise ratio resulting from the higher ion trapping capacity of the RIT. The demonstrated capabilities of the RIT include tandem mass spectrometry, a mass resolution in excess of 1000, and a mass/charge range of 650 Th, all in a simple structure that is only 3.5 cm(3) in internal volume.     

Coaxial ion trap mass spectrometer: concentric toroidal and quadrupolar trapping regions

We present the design and results for a new radio-frequency ion trap mass analyzer, the coaxial ion trap, in which both toroidal and quadrupolar trapping regions are created simultaneously. The device is composed of two parallel ceramic plates, the facing surfaces of which are lithographically patterned with concentric metal rings and covered with a thin film of germanium. Experiments demonstrate that ions can be trapped in either region, transferred from the toroidal to the quadrupolar region, and mass-selectively ejected from the quadrupolar region to a detector. Ions trapped in the toroidal region can be transferred to the quadrupole region using an applied ac signal in the radial direction, although it appears that the mechanism of this transfer does not involve resonance with the ion secular frequency, and the process is not mass selective. Ions in the quadrupole trapping region are mass analyzed using dipole resonant ejection. Multiple transfer steps and mass analysis scans are possible on a single population of ions, as from a single ionization/trapping event. The device demonstrates better mass resolving power than the radially ejecting halo ion trap and better sensitivity than the planar quadrupole ion trap.     

Ion funnel trap interface for orthogonal time-of-flight mass spectrometry

A combined electrodynamic ion funnel and ion trap coupled to an orthogonal acceleration (oa)-time-of-flight mass spectrometer was developed and characterized. The ion trap was incorporated through the use of added terminal electrodynamic ion funnel electrodes enabling control over the axial dc gradient in the trap section. The ion trap operates efficiently at a pressure of approximately 1 Torr, and measurements indicate a maximum charge capacity of approximately 3 x 10(7) charges. An order of magnitude increase in sensitivity was observed in the analysis of low concentration peptides mixtures with orthogonal acceleration (oa)-time-of-flight mass spectrometry (oa-TOF MS) in the trapping mode as compared to the continuous regime. A signal increase in the trapping mode was accompanied by reduction in the chemical background, due to more efficient desolvation of, for example, solvent related clusters. Controlling the ion trap ejection time was found to result in efficient removal of singly charged species and improving signal-to-noise ratio (S/N) for the multiply charged analytes.     

Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra

High-resolution and high-accuracy Fourier transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5-3.0-fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS spectra which are then combined into a single concatenated file and submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations.     

An interface with a linear quadrupole ion guide for an electrospray-ion trap mass spectrometer system

A new ion sampling interface for an electrospray ionization 3D ion trap mass spectrometer system is described. The interface uses linear rf quadrupoles as ion guides and ion traps to enhance the performance of the 3D trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. A resolution of ejection of up to 254 is demonstrated for protonated reserpine ions (m/z 609.3). A composite waveform with a notch in frequency space is used to eject a wide range of matrix ions and to isolate trace analyte ions in a linear quadrupole before ions are injected into the 3D trap. This is useful to overcome space charge problems in the 3D trap caused by excess matrix ions. For trace reserpine in a 500-fold molar excess of poly(propylene glycol) (PPG), it is demonstrated that the resolution and sensitivity of the 3D trap can be increased dramatically with ejection of the excess PPG matrix ions. In comparison to ejection of matrix ions in the 3D trap with a similar broad-band waveform, a 5-fold increase in sensitivity with a 7 times shorter acquisition time was achieved.     

MSNovo: a dynamic programming algorithm for de novo peptide sequencing via tandem mass spectrometry

Tandem mass spectrometry (MS/MS) has become the experimental method of choice for high-throughput proteomics-based biological discovery. The two primary ways of analyzing MS/MS data are database search and de novo sequencing. In this paper, we present a new approach to peptide de novo sequencing, called MSNovo, which has the following advanced features. (1) It works on data generated from both LCQ and LTQ mass spectrometers and interprets singly, doubly, and triply charged ions. (2) It integrates a new probabilistic scoring function with a mass array-based dynamic programming algorithm. The simplicity of the scoring function, with only 6-10 parameters to be trained, avoids the problem of overfitting and allows MSNovo to be adopted for other machines and data sets easily. The mass array data structure explicitly encodes all possible peptides and allows the dynamic programming algorithm to find the best peptide. (3) Compared to existing programs, MSNovo predicts peptides as well as sequence tags with a higher accuracy, which is important for those applications that search protein databases using the de novo sequencing results. More specifically, we show that MSNovo outperforms other programs on various ESI ion trap data. We also show that for high-resolution data the performance of MSNovo improves significantly. Supporting Information, executable files and data sets can be found at http://msms.usc.edu/supplementary/msnovo.     

Selective detection of glycopeptides on ion trap mass spectrometers

Generation of carbohydrate-specific marker ions during LC-ESMS of digested glycoproteins has been demonstrated to be a highly selective and sensitive approach for detection of glycopeptides. In principle, any mass spectrometer can produce and selectively detect carbohydrate marker ions provided that the instrument is capable of collisional excitation in the region prior to the first mass analyzer sufficient to form abundant oxonium ions. This approach has yet to be demonstrated on 3D ion trap mass spectrometers, which have become widely used for proteomic applications. Here we report the successful development and optimization of carbohydrate marker ion detection on a LCQ Deca 3D ion trap utilizing this scan function. Human alpha-1 acid glycoprotein and a therapeutic monoclonal antibody were chosen to illustrate this methodology. Marker ion detection during LC-ESMS facilitated collection of glycopeptide-containing fractions. Analysis of the glycopeptides in these fractions by MS identified the specific glycosylation sites and enabled the prediction of the family of glycoforms at each attachment site. Using these optimized conditions, marker ion detection and glycopeptide analysis could be achieved with as little as 10 pmol of a glycoprotein.     

Halo ion trap mass spectrometer

We describe a novel radio frequency ion trap mass analyzer based on toroidal trapping geometry and microfabrication technology. The device, called the halo ion trap, consists of two parallel ceramic plates, the facing surfaces of which are imprinted with sets of concentric ring electrodes. Radii of the imprinted rings range from 5 to 12 mm, and the spacing between the plates is 4 mm. Unlike conventional ion traps, in which hyperbolic metal electrodes establish equipotential boundary conditions, electric fields in the halo ion trap are established by applying different radio frequency potentials to each ring. The potential on each ring can be independently optimized to provide the best trapping field. The halo ion trap features an open structure, allowing easy access for in situ ionization. The toroidal geometry provides a large trapping and analyzing volume, increasing the number of ions that can be stored and reducing the effects of space-charge on mass analysis. Preliminary mass spectra show resolution (m/Deltam) of 60-75 when the trap is operated at 1.9 MHz and 500 Vp-p.     

On-line 1D and 2D porous layer open tubular/LC-ESI-MS using 10-microm-i.d. poly(styrene-divinylbenzene) columns for ultrasensitive proteomic analysis

Following on our recent work, on-line one-dimensional (1D) and two-dimensional (2D) porous layer open tubular/liquid chromatography-electrospray ionization-mass spectrometry (PLOT/LC-ESI-MS) platforms using 3.2 mx10 microm i.d. poly(styrene-divinylbenzene) (PS-DVB) PLOT columns have been developed to provide robust, high-performance, and ultrasensitive proteomic analysis. With the use of a PicoClear tee, the dead volume connection between a 50 microm i.d. PS-DVB monolithic micro-SPE column and the PLOT column was minimized. The micro-SPE/PLOT column assembly provided a separation performance similar to that obtained with direct injection onto the PLOT column at a mobile phase flow rate of 20 nL/min. The trace analysis potential of the platform was evaluated using an in-gel tryptic digest sample of a gel fraction (15-40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, approximately 2.5 ng of protein in 2 microL of solution, an amount corresponding to 20 SiHa cells, was subjected to on-line micro-SPE-PLOT/LC-ESI-MS/MS analysis using a linear ion trap MS. A total of 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate of less than 1%. The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to approximately 45 ng of protein, an amount corresponding to 350 SiHa cells. In comparison, only 338 peptides and 231 unique proteins were identified (false positive rate again less than 1%) from 750 ng of protein from the identical gel fraction, an amount corresponding to 6000 SiHa cells, using a typical 15 cmx75 microm i.d. packed capillary column. The greater sensitivity, higher recovery, and higher resolving power of the PLOT column resulted in the increased number of identifications from only approximately 5% of the injected sample amount. The resolving power of the micro-SPE/PLOT assembly was further extended by 2D chromatography via combination of the high-efficiency reversed-phase PLOT column with strong cation-exchange chromatography (SCX). As an example, 1071 peptides associated with 536 unique proteins were identified from 75 ng of protein from the same gel fraction, an amount corresponding to 600 cells, using five ion-exchange fractions in on-line 2D SCX-PLOT/LC-MS. The 2D system, implemented in an automated format, led to simple and robust operation for proteomic analysis. These promising results demonstrate the potential of the PLOT column for ultratrace analysis.     

A parallel miniature cylindrical ion trap array

A small mass spectrometer array is described in which each element is a cylindrical ion trap (CIT). The array contains four CITs, each having an inner radius of 2.5 mm, arranged in parallel and operated using a single electronics system under common conditions for trapping and mass analysis. By using an array of identically sized traps, higher ion capacity can be achieved than with a single miniature CIT, but the advantage of lower power and voltage requirements associated with the smaller ion trap is maintained. Overall signal intensity of the array of four traps is compared with that of a two-element CIT array to demonstrate the increased ion capacity of larger arrays. Resolution for m-dichlorobenzene is shown to be approximately 180 (full width at half-maximum), with no significant loss in resolution as a result of using multiple CITs. The detection of 5 x 10(-9) Torr partial pressure of krypton in argon with a signal-to-noise ratio of approximately 30 for the most abundant isotope is shown, demonstrating the applicability of the device for process gas monitoring. Also, a preliminary spectrum from a 10-CIT array with each element having an inner radius of 1.5 mm is reported.     

Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.     

Hole Surface Trapping in CdSe Nanocrystals: Dynamics, Rate Fluctuations, and Implications for Blinking

Carrier trapping is one of the main sources of performance degradation in nanocrystal-based devices. Yet the dynamics of this process is still unclear. We present a comprehensive investigation into the efficiency of hole transfer to a variety of trap sites located on the surface of the core or the shell or at the core/shell interface in CdSe nanocrystals with both organic and inorganic passivation, using the atomistic semiempirical pseudopotential approach. We separate the contribution of coupling strength and energetics in different systems and trap configurations, obtaining useful general guidelines for trapping rate engineering. We find that trapping can be extremely efficient in core-only systems, with trapping times orders of magnitude faster than radiative recombination. The presence of an inorganic shell can instead bring the trapping rates well below the typical radiative recombination rates observed in these systems.     

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.     

Relative quantification of stable isotope labeled peptides using a linear ion trap-Orbitrap hybrid mass spectrometer

The quantitative analysis of complex biological samples has emerged as a key research area in the field of proteomics. Although quantitative proteomic experiments remain challenging, these strategies have been greatly facilitated by the development of newer high-performance mass spectrometers. In this work, we have evaluated the use of the LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer, for quantitative analyses. By analyzing a range of yeast protein standards, we found that the high mass accuracy, high resolution, large ion capacity, and large dynamic range of the LTQ-Orbitrap led to as much as a 4-5-fold improvement in the number and quality of the peptide ratio measurements compared to similar analyses done on the LTQ. We also successfully quantified protein expression differences that occur in metabolically labeled rat synapses during brain development to further demonstrate the suitability of the LTQ-Orbitrap for the comparative analysis of complex tissue samples.     

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 parking during ion/ion reactions in electrodynamic ion traps.

Under appropriate ion density conditions, it is possible to selectively inhibit rates of ion/ion reactions in a quadrupole ion trap via the application of oscillatory voltages to one or more electrodes of the ion trap. The phenomenon is demonstrated using dipolar resonance excitation applied to the end-cap electrodes of a three-dimensional quadrupole ion trap. The application of a resonance excitation voltage tuned to inhibit the ion/ion reaction rate of a specific range of ion mass-to-charge ratios is referred to as "ion parking". The bases for rate inhibition are (i) an increase in the relative velocity of the ion/ion reaction pair, which reduces the cross section for ion/ion capture and, at least in some cases, (ii) reduction in the time of physical overlap of positively charged and negatively charged ion clouds. The efficiency and specificity of the ion parking experiment is highly dependent upon ion densities, trapping conditions, ion charge states, and resonance excitation conditions. The ion parking experiment is illustrated herein along with applications to the concentration of ions originally present over a range of charge states into a selected charge state and in the selection of a particular ion from a set of ions derived from a simple protein mixture.     

Handheld rectilinear ion trap mass spectrometer

A shoebox-sized, 10-kg, handheld mass spectrometer, Mini 10, based on a rectilinear ion trap mass analyzer has been designed, built, and characterized. This instrument has evolved from a decade-long experimental and simulation program in mass spectrometer miniaturization. The rectilinear ion trap has a simplified geometry and high trapping capacity, and when used with a miniature and ruggedized pumping system, it allows chemical analysis while the instrument is being carried. Compact electronics, including an air core RF drive coil, were developed to control the instrument and to record mass spectra. The instrument runs on battery power, consuming less than 70 W, similar to a laptop computer. Wired and wireless networking capabilities are implemented. The instrument gives unit resolution and a mass range of over m/z 500. Tandem mass spectrometry capabilities are implemented using collision-induced dissociation, and they are used to provide confirmation of chemical structure during in situ analysis. Continuous monitoring of air and solution samples is demonstrated, and a limit of detection of 50 ppb was obtained for toluene vapor in air and for an aqueous naphthalene solution using membrane sample introduction.     

Electron capture dissociation in a digital ion trap mass spectrometer

Electron capture dissociation was implemented in a digital ion trap without using any magnetic field to focus the electrons. Since rectangular waveforms are employed in the DIT for both trapping and dipole excitation, electrons can be injected into the trap when the electric field is constant. Following deceleration, electrons reach the precursor ion cloud. The fragment ions produced by interactions with the electron beam are subsequently analyzed by resonant ejection. [Glu(1)]-Fibrinopeptide B and substance P were used to evaluate the performance of the current design. Fragmentation efficiency of 5.5% was observed for substance P peptide ions. Additionally, analysis of the monophosphorylated peptide FQ[pS]EEQQQTEDELQDK shows that in the resulting c- and z-type ions, the phosphate group is retained on the phophoserine residue, providing information on which amino acid residue the modification is located.