Abstract:
One aspect of the system provides the use of a laser with a mass spectrometer. Another aspect of the present application employs a laser emitting a pulse of less than one picosecond duration into an ion-trap mass spectrometer. In yet another aspect of the present application, a femtosecond laser beam pulse is emitted upon an ionized specimen to remove at least one electron therefrom.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application Claims the benefit of U.S. Provisional Application No. 61/114,809, filed on Nov. 14, 2008, which is incorporated by reference herein. 
     
    
     STATEMENT OF GOVERNMENT INTEREST 
       [0002]    A portion of this invention was made with U.S. government support under Grant Nos. CHE-0547940 and CHE-0647901 from the National Science Foundation. The U.S. government may have certain rights in this invention. 
     
    
     BACKGROUND AND SUMMARY 
       [0003]    This application relates generally to mass spectrometry and more particularly to an ultrafast laser system for biological mass spectrometry. 
         [0004]    Over the past decade, mass spectrometry (“MS”) has become a key analytical tool for analyzing proteins and metabolites. MS has been used to identify post-translational modifications (“PTMs”) of proteins, which are in some cases the signature of aging processes and malignant disease, making them valuable markers for medical diagnosis. Typically, complex protein mixtures or individual proteins resolved by electrophoretic or chromatographic methods have been traditionally subjected to proteolysis, and then the resultant peptide mixtures were introduced to the mass spectrometer by on-line chromatography. Peptide sequence information was then obtained via subjecting individual ions to fragmentation by collision-induced dissociation (“CID”) tandem mass spectrometry (“MS/MS”). Protein identification was then achieved by database analysis using sophisticated search algorithms (e.g., SEQUEST, Mascot), to correlate the uninterpreted peptide MS/MS spectra with simulated (predicted) product ion spectra derived from peptides of the same mass contained in the available databases. However, the generally limited ability to selectively control or direct the fragmentation reactions of peptide ions during CID-MS/MS towards the formation of structurally informative ‘sequence’ ions (i.e., those resulting from amide peptide bond cleavages) or ‘non-sequence’ ions (i.e., those resulting from cleavage of amino acid side chains that are characteristic of the presence of post translational modifications), placed significant limitations on the application of mass spectrometry and associated methodologies for comprehensive proteome analysis. Recently, several groups have begun to explore the use of laser photo-induced dissociation (“PID”) to access alternative or complementary fragmentation pathways to those observed by conventional collision-induced dissociation. However, these approaches typically did not have bond-selective control over the site of energy absorption from the laser pulse, due to rapid intramolecular vibrational relaxation that occurred prior to bond cleavage, and typically required the presence of a chromophore that was able to absorb energy at the wavelength of the laser to induce fragmentation. 
         [0005]    The application of tandem mass spectrometry (“MS/MS”) methods to the identification and characterization of proteolytically derived peptide ions has underpinned the emergent field of proteomics. However, the ability of these conventional approaches to generate sufficient product ions from which the sequence of an unknown peptide can be determined, or to unambiguously characterize the specific site(s) of post-translational modifications within these peptides, was highly dependant on the specific method employed for ion activation, as well as the sequence and charge state of the precursor ion selected for analysis. In practice, collision induced dissociation, whereby energy deposition occurs through ion-molecule collisions followed by internal vibrational energy redistribution prior to dissociation, often resulted in incomplete backbone fragmentation, or the dominant loss of labile groups from side chains containing post-translational modifications such as phosphorylation, particularly for peptides observed at low charge states. Thus, there has been great interest in the development of alternate activation methods, such as surface induced dissociation (“SID”), infrared multiphoton dissociation (“IRMPD”), ultraviolet photodissociation (“UVPD”), electron capture and electron transfer dissociation (“ECD” and “ETD”) and metastable atom dissociation, that yield greater sequence information, and that provide selective control over the fragmentation chemistry independently of the identity of the precursor ion. However, each of these methods suffers from certain limitations. For example, ECD and ETD are applicable only to the analysis of multiply-charged precursor ions, while IRMPD and UVPD efficiencies are dependant on the presence of a suitable chromophore for photon absorption. 
         [0006]    In accordance with the present application, one aspect of the system provides a laser and a mass spectrometer. Another aspect of the present application employs a laser emitting a laser beam pulse duration of less than one picosecond into an ion-trap mass spectrometer. A further aspect of the present application provides entrance and exit holes in a mass spectrometer for a laser beam pulse passing therethrough, which advantageously reduces undesired surface charges otherwise possible from misalignment within the mass spectrometer. In yet another aspect of the present application, a femtosecond laser beam pulse causes the ultrafast loss of an election from the charged ions for optional further fragmentation and more detailed mass spectrometry analysis. Another aspect of the present application uses electrospray with mass spectrometry and a shaped laser beam pulse having a duration of less than one picosecond. In still another aspect of the present application, Multiphoton Intrapulse Interference Phase Scan procedures are used to characterize and compensate for undesired characteristics in a laser beam pulse used with an ion-trap mass spectrometer. An additional aspect of the present application includes software instructions which assist in determining whether desired mass spectra information has been obtained, and if not, isolating product ions and then causing another ionization and/or fragmentation process to occur. A method of using a laser system for biological mass spectrometry is also provided. Another method employs emitting a shaped laser pulse at an ionized specimen, further ionizing the ionized specimen by removing at least one electron, isolating the ionized specimen, and then using another supplemental activation step including at least one of fs-LID, CID, SID, IRMPD, UVPD, ECD ETD, Post-Source Decay (“PSD”), Electron Ionization Dissociation (“EID”), Electronic Excitation Dissociation (“EED”), Electron Detachment Dissociation (“EDD”), and/or Metastable Atom-activated Dissociation (“MAD”), in the same equipment. 
         [0007]    In order to overcome limitations of conventional devices, the present application provides an advantageous approach to protonated peptide sequence analysis and characterization, involving the use of ultrashort laser pulses for nonergodic energy deposition and multistage dissociation in a quadrupole ion trap mass spectrometer. In one aspect of the present application, peptide solutions in methanol/water/acetic acid are introduced to the mass spectrometer by electrospray ionization, then selected precursor ions are isolated and subjected to MS/MS and MS 3  by fs-LID or CID. 
         [0008]    The present system significantly improves the structural analysis of modified proteins by the introduction of a femtosecond laser into an ion-trap mass spectrometer. The goal is to take advantage of ultrafast activation, i.e. faster than intramolecular energy redistribution, in order to control the ionization and fragmentation processes. Pulse shaping, in this context, provides in-situ selective fragmentation of specific bonds within a peptide. Binary shaped laser pulses are highly effective in controlling the fragmentation of volatile compounds, and when coupled to an ionization source compatible with the introduction of biomolecules into the gas-phase, provides hitherto unavailable structural information for protein sequencing (proteomics), metabolite recognition (metabolomics), lipid characterization (lipidomics) and target-binding recognition such as protein-ligand, and protein-protein interactions (drug design). A shaped femtosecond laser of the present invention can control the ionization and dissociation processes of isolated ions in the gas-phase due to its ability to deliver energy in a timescale faster than intramolecular energy relaxation. This improves two aspects of biological mass spectrometry: Providing greater sequence coverage than conventional methods such as collision induced dissociation, and improving the analysis of modified proteins by avoiding loss or scrambling of the modification group. The acquisition of reproducible dissociation in the mass spectrometer harnesses the ability to deliver transform limited pulses, i.e., without spectral phase distortions, at the ion-packet within the ion trap of a mass spectrometer. 
         [0009]    Simple fragmentation of ions by using short wavelength laser sources in the UV and sometimes in the near UV (400 nm) is well known. For these fragmentation processes to occur it is important for the ion of interest to have at least some portion of its molecular structure include a chromophore or region which by itself has an absorption in the UV-Vis wavelength. In such cases absorption of one or two photons deposits energy in the molecule leads to bond dissociation. The amount of energy will equal that of one or at most two photons which in this case will be less than 10 eV. The drawback to this approach is that short-wavelength laser wavelengths are difficult to generate especially with high energy per pulse. In addition, the molecule or ion must absorb the incident wavelength. It would be advantageous to use an approach that can be used with all molecules and ions without requiring that they absorb the incident wavelength. This approach becomes accessible with ultrafast (preferably less than 1 picosecond and more preferably less than 60 femtosecond) laser pulses of the present disclosure, especially those that have longer wavelengths (from near-infra red 700 nm and longer in the infrared 1 to 2 μm). 
         [0010]    The present system&#39;s use of ultrafast laser pulses opens a new approach to ion activation. The interaction of an ultrafast laser pulse and an ion is very different from that of a nanosecond laser pulse, especially when the photon energy is much smaller than the ionization potential. In general, ionization of a neutral molecule or further ionization of a trapped ion requires 7-9 eV of energy. This energy can be provided through a nonlinear optical interaction between a long wavelength laser (with energy much smaller than that required for ionization) and the molecule. One may loosely divide the character of the intense-laser nonlinear optical ionization into (a) multi-photon ionization, (b) tunneling ionization and (c) over-the barrier ionization. A Keldysh parameter is used for the classification. A free electron in a laser field makes an oscillating motion at the frequency of the laser. The quiver energy or ponderomotive energy is given by 
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         [0000]    and ω is the angular frequency of the laser electric field, or alternatively I 0  and λ are the intensity and the wavelength of the laser field. The Keldysh parameter is proportional to the ratio between the binding energy, E B , of the electron and the ponderomotive energy. It is defined as 
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         [0000]    and it is noteworthy that the Keldysh parameter is inversely proportional to the wavelength of the laser. 
         [0011]    γ as a function of the intensity and wavelength is then calculated. The multi-photon regime corresponds to the condition where γ&gt;1. In the tunneling regime, scattering with the nuclear center is not important. Instead, the potential barrier formed by the core of the atom or molecule and the electric field of the laser becomes small enough for tunneling to become possible. The electron is pulled off in a field ionization process. Nevertheless, there is a difference between a static field and an oscillating field of the same magnitude. In a static field, a tunneling current will always build up. In an oscillating field, a starting tunneling current is pushed back in the next half cycle, unless it is fast enough to reach the other side of the barrier. It can be shown that the Keldysh parameter is also a measure of the ratio between the laser period and the tunneling time. Thus, when γ≈1 or smaller, the laser field can be treated as quasi-static. Generally, the tunneling formula of Amosov, Delone and Krainov (ADK theory) is considered to be a good approximation to the ionization rate. 
         [0012]    For γ&lt;1, ionization is in the over-the-barrier regime. In this case, the electron can escape classically from the potential well. There is, however, no sudden step in the ionization rate at the threshold for over-the-barrier ionization. Instead, the ionization rate continues to grow smoothly and continuously with increasing laser intensity. 
         [0013]    Given a certain laser in the laboratory, a minimum value of laser intensity will be required to observe the highly non-linear process involving over-the-barrier ionization; this is the so-called appearance intensity. At somewhat higher laser intensity, the saturation intensity, the ionization rate will have increased so much that the process saturates, i.e. the ionization probability approaches 1. For femtosecond lasers, the over-the-barrier regime is significant. At the classical threshold, the ionization lifetime, i.e. the inverse of the ionization rate, is of the order of 10-100 fs. 
         [0014]    Therefore, activation of trapped ions is best achieved by using ultrafast long-wavelength pulses in the present system rather than by using conventional UV-Vis lasers (although certain of the present Claims may not be so limited). The activation proceeds through over-the-barrier ionization. The ion of charge n is ionized to produce a radical ion of charge n+1. The newly created ion can also acquire additional energy which leads to fragmentation. The processes that become available with ultrafast lasers with long wavelengths can be used for (i) altering the charge of trapped ions via removal of electrons and to (ii) fragment trapped ions in a time scale that is much faster than intramolecular vibrational relaxation. Fast fragmentation of ions is desirable when the ions have both strong and weak chemical bonds. Unlike slow fragmentation processes like collision induced dissociation in which there is a thermal or statistical distribution of energy, ultrafast fragmentation prevents the redistribution of energy. In slow fragmentation the weak bonds break preferentially and strong bonds cannot be broken. In contrast, in the fast fragmentation of the present system, strong bonds are broken and weak bonds are left intact. This latter case is important for the analysis of post-translational modifications (“PTM”) of proteins. PTM&#39;s have been linked to specific diseases, to aging and as markers for stress. Therefore PTM analysis is beneficial for marker elucidation, for diagnostic purposes, and for monitoring the progression of a disease. It is also noteworthy that over-the-barrier ionization of polyatomic molecules becomes more efficient when circularly polarized femtosecond lasers are used. 
         [0015]    Pulse characterization and compression are preferably employed with another aspect of the present invention. With the pulse shaper, the pulse duration is controlled and the pulses are tailored to explore the parameter space that provides the desired level of bond dissociation. Additional advantages and features of the present invention will become apparent from the following description and appended Claims, taken in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1  is a diagrammatic view showing a laser system and a modified, three dimensional, fs-LID ion-trap mass spectrometer used in the system of the present invention; 
           [0017]      FIG. 2  shows an expected Ti:Sapphire laser spectrum, illustrating the broad bandwidth of the ultrafast laser source, for the present invention system; 
           [0018]      FIG. 3  shows expected results at (A) fs-LID of the [M+H] +  precursor ion of angiotensin II, and at (B) CID MS 3  of the [M+H] 2+ • photoionization product from panel A, for the present system; 
           [0019]      FIG. 4  shows expected results at (A) fs-LID of the [M+H] +  precursor ion of GAILpTGAILK, and at (B) CID MS 3  of the [M+H] 2+ • photoionization product from panel A, for the present invention system; 
           [0020]      FIG. 5  shows expected CID-MS/MS of the (A) [M+H] + , (B) [M+2H] 2+  and (C) [M+3H] 3+  precursor ions of angiotensin II, for the present invention system; 
           [0021]      FIG. 6  shows expected high resolution zoomscan spectra of (A) the isolated [M+H] 2+ • photoionization product ion of angiotensin II from  FIGS. 3A , and  3 B the isolated [M+2H] 2+  precursor ion of angiotensin II, for the present invention system; 
           [0022]      FIG. 7  shows expected fsLID-MS 3  (q=0.25) of the [M+H] 2+  photoionization product of angiotensin II from  FIG. 3A , for the present invention system; 
           [0023]      FIG. 8  shows expected CID-MS/MS of (A) the [M+H] +  and (B) [M+2H] 2+  precursor ions of the model synthetic phosphothreonine containing peptide GAILpTGAILK (pTK), for the present invention system; 
           [0024]      FIG. 9  shows expected fsLID-MS 3  (q=0.25) of the [M+H] 2+ • photoionization product of the model synthetic phosphothreonine containing peptide GAILpTGAILK (pTK) from  FIG. 4A , for the present invention system; 
           [0025]      FIG. 10  shows expected fsLID-MS/MS of the (A) [M+2H] 2+  and (B) [M+2H] 2+  precursor ions of angiotensin II, for the present invention system; 
           [0026]      FIG. 11  shows expected fsLID-MS/MS of the [M+2H] 2+  precursor ion of the model synthetic phosphothreonine containing peptide GAILpTGAILK (pTK), for the present invention system; 
           [0027]      FIG. 12  is a plot showing expected results on the effect of binary phase shaping on the photodissociation of pyridine for the present invention system; 
           [0028]      FIG. 13  shows on the left, conventional MS data for ortho- and para-xylene showing there is little or no difference in their spectra, and on the right, by using fs-dissociation o- and p-xylene are expected to be easily identified for the present invention system. Using binary phase shaping, the dissociation is much more pronounced. The expected data correspond to the ratio M + /T +  and given are the histogram from 128 expected measurements; 
           [0029]      FIG. 14  shows at (A) an expected CID spectrum of angiotensin II, at (B) an expected fs-LID spectrum under the same conditions showing a much greater degree of sequence specific bond cleavage, and generating a doubly charged radical molecular ion product, and at (C) an expected MS 3  CID spectrum of the fs-LID doubly charged radical ion for the present invention system; 
           [0030]      FIG. 15  shows at (A) an expected CID spectrum of pTK showing phosphate loss as the main product, at (B) an expected fs-LID spectrum under the same conditions showing a much greater degree of sequence specific bond cleavage with much fewer loss of the phosphate group and generating the doubly ionized molecular ion-radical, and at (C) an expected MS 3  CID spectrum of the fs-LID doubly charged ion-radical for the present invention system; 
           [0031]      FIG. 16  is a diagrammatic view showing a single pass cavity in a mass spectrometer of the present invention system; 
           [0032]      FIG. 17  is a diagrammatic view showing a double pass cavity in a mass spectrometer of the present invention system; 
           [0033]      FIG. 18  is a diagrammatic view showing a multipass cavity in a mass spectrometer of the present invention system; 
           [0034]      FIG. 19  is a diagrammatic view showing a laser system and a linear, fs-LID ion-trap mass spectrometer used in the system of the present invention; and 
           [0035]      FIG. 20  is a software flow chart for the present invention system. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0036]    Referring to  FIG. 1 , a preferred embodiment of the ultrafast laser system  31  of the present invention includes a 3D ion-trap mass spectrometer  33  (model LCQ Deca XP Plus, Thermo Scientific, San Jose, Calif.) modified using the following specific conditions: a ½″ hole is drilled through the left hand side of a vacuum manifold  35  at a 60° downward angle, in line with the center of a ring electrode  37  of an ion-trap  39 , and an aluminum conflat nipple is welded to the manifold. A series of ½″ KwikFlange components are used to construct a vacuum-sealed entrance port  41  for a laser pulse  43 , capped with a 1″ diameter fused silica window  45 . A 5 mm hole  47  is drilled through ring electrode  37  of the ion-trap, and quartz spacers on either side of the ring electrode are notched to allow clear passage of the laser pulse through the ion-trap. A silver mirror  49 , mounted on a custom-cut aluminum block, is then fixed to vacuum manifold  35  on the far side of the trap to direct laser beam pulse  43  out a ½″ diameter hole (capped with another 1″ diameter fused silica window  51 ) drilled through the back of the vacuum manifold. The original helium gas flow regulator is removed and replaced with a manual flow controller (Porter, model VCD 1000) to allow for re-optimization of the helium gas pressure inside the ion-trap to account for increased leakage through the newly-drilled holes. Optimal performance (i.e., similar sensitivity, mass resolution and collision induced dissociation efficiency to that obtained in the unmodified instrument) should be achieved when the ion gauge pressure is between 2.5×10 −5  and 3.0×10 −5  Torr. 
         [0037]    An electrospray ionization (“ESI”), matrix assisted laser desorption ionization (“MALDI”), desorption electrospray ionization (“DESI”), or other precursor ionized specimen source  53  is provided. For example, a syringe pump containing the ionized specimen is mounted to a receptacle  55  of mass spectrometer  33 , adjacent ion transfer optics  57 . Entrance and exit endcap electrodes,  59  and  61 , respectively, are located between optics  57  and ring electrode  37 . Furthermore, a main RF power supply  63  is electrically connected to ring electrode  37  for trapping the ions, and a supplemental RF frequency synthesizer  65  is electrically connected to endcap electrodes  59  and  61  for isolating precursor ions and/or for CID processing. A mass spectrum detector  67  is located adjacent exit endcap electrode  61 , which sends sensed mass charge information to a computer controller  69  electrically connected thereto. Frequency synthesizer  65 , power supply  63  and electrospray source  53  are also directly or indirectly electrically connected to and automatically controlled by computer  69 . 
         [0038]    The output of a regeneratively amplified Ti:Al 2 O 3  laser (Spitfire—Spectra Physics, Mountain View, Calif.), seeded with a broadband Ti:Al 2 O 3  oscillator  81  (KM Labs, Boulder, Colo.) operated at a 1 kHz repetition rate, with a 35 fs pulse duration and a 26 nm bandwidth centered at 800 nm, is attenuated to 300 μJ/pulse and focused into the mass spectrometer using a periscope  83  and an optic member  85 , such as a f=400 mm lens. An iris optic member  86  and filter  87  are also employed. Spectral phase distortions are measured at the sample and compensated using a MIIPS Box Pulse Shaper  88  from Biophotonics Solutions, Inc. (East Lansing, Mich.), resulting in transform limited pulses passing through an amplifier  89  as shown in  FIG. 2 . Such a Multiphoton Intrapulse Interference Phase Scan (“MIIPS”) system is disclosed in PCT Patent Publication Nos. WO 2006/088841, entitled “Ultra-Fast Laser System,” and WO 2007/008615, entitled “Control System and Apparatus for Use with Ultra-Fast Laser,” both of which were invented by M. Dantus et al., and are incorporated by reference herein. Alignment of the laser beam with the ion packet is fine tuned using a pair of adjustable mirrors on the periscope, and by monitoring the laser output through the exit hole located at the back of the MS instrument to ensure unobstructed passage through the closed vacuum system. 
         [0039]    The laser is triggered using a Uniblitz LS-series shutter  91  (Rochester, N.Y.) controlled from the advanced Diagnostics menu within the Tune Plus window of Xcalibur software to generate a TTL output signal from TP_ 15  of the mass spectrometer to the shutter controller during the ion activation time period of a specified MS/MS or MS 3  experiment. Under the tab labeled ‘Triggers’ within the Diagnostics menu, the trigger location is set to ‘activation’ and the scan position is set to ‘0’ to generate a TTL pulse at the beginning of the activation period of an fs-LID-MS/MS experiment, but not during subsequent activation periods, or at to generate a TTL pulse at the beginning of each activation time period for an MS 3  experiment (i.e, fs-LID-MS/MS/fs-LID-MS 3 ). Alternately, a CID-MS/MS/fs-LID-MS 3  experiment may be performed by setting the scan position to ‘1’. The ‘open’ time for the shutter could be set independently of the MS/MS or MS 3  activation time period. For this preferred embodiment, however, the shutter timing and the MS/MS or MS 3  activation time periods are identical. 
         [0040]    Angiotensin II (DRVYIHPF) is purchased from Sigma Aldrich and used without further purification. The model synthetic phosphopeptide GAILpTGAILK (pTK) is prepared and samples (10 μM) dissolved in methanol/water/acetic acid (50:50:1) are introduced to the mass spectrometer by electrospray ionization using the syringe pump operated at a flow rate of 3 μL/min, a spray voltage of 4.0 kV, a heated capillary temp of 200° C., a tube lens offset of 40 V and a capillary voltage of 35 V. 
         [0041]    Ion-trap fs-Laser induced Ionization/Dissociation (fs-LID) and collision induced dissociation (“CID”) MS/MS and MS 3  experiments are performed on mass selected protonated precursor ions, using an isolation width of 4-6 m/z, and an activation q-value of 0.17, unless otherwise stated. In order to obtain product ion spectra with good signal-to-noise, fs-LID MS/MS and MS 3  spectra are collected using an irradiation period of 200 msec. CID MS/MS and MS 3  spectra are collected using an activation time of 30 msec. The fs-LID spectra shown are the expected average of 500 scans, while CID spectra are the expected average of 200 scans (3 microscans/scan). All spectra are shown in profile mode and a 5 point Gaussian smooth is applied to all spectra. Repeated analysis of expected individual samples results in less than 5% variation in relative product ion abundances. For high resolution zoomscans of isolated [M+2H] 2+  and [M+H] 2+•  ions of angiotensin II, the automatic gain control (AGC) target is set to 1×10 6 . 
         [0042]    fs-LID of the [M+H] +  precursor ion of angiotensin II ( FIG. 3A ) should yield  23  of the  42  possible a-, b-, c-, x-, y- and z-type ‘sequence’ product ions, from which 100% sequence coverage is obtained. In comparison, the CID spectrum obtained from the same precursor ion is dominated by selective cleavage at the C-terminal side of the aspartic acid residue to yield the y 7  ion ( FIG. 5 ), and should yield only (12 of 42 possible sequence ions). 
         [0043]    Notably, an odd electron doubly charged ([M+H] 2+ •) product ion should be observed in  FIGS. 3A and 6 , via photoionization of the even electron singly protonated precursor. [M+H] 2+ •ions have previously been produced by electron ionization or as Penning ionization products following metastable ion activation. However, these ions have not previously been observed via conventional photoionization techniques, and their involvement in the dissociation pathways responsible for the formation of sequence type product ions have not previously been reported. CID-MS 3  ( FIG. 3B ) and fs-LID MS 3  ( FIG. 7 ) reveal that the majority of the product ions to be observed in  FIG. 3  are indeed formed from this species. Other product ions formed via the losses of p-quinomethide (−106) and COOH• (−45), indicative of specific side chain functional groups in the peptide are also observable in  FIG. 3A . 
         [0044]    To assess the utility of fs-LID for the characterization of peptide post-translational modifications, the fragmentation reactions of the [M+H] +  precursor ion from a model synthetic phosphopeptide GAILpTGAILK (pTK) 4  is examined. CID MS/MS of this peptide ( FIG. 8 ) should result in the dominant loss of H 3 PO 4 , precluding the ability to assign the site of phosphorylation. However, it can be seen from  FIG. 4  that 100% sequence coverage is obtained upon fs-LID (21 of the 54 possible a-, b-, c-, x-, y- and z-type ‘sequence’ product ions), with only minimal loss of the phosphate group. Similar to that discussed above for angiotensin II, an [M+H] 2+ • product ion is also observed for the pTK peptide. CID-MS 3  and fs-LID MS 3  ( FIG. 4B  and  FIG. 9 , respectively) again reveals that the majority of the observed product ions in  FIG. 4A  are formed via this product. The fs-LID technique is also generally applicable to the analysis of multiply protonated precursor ions, as shown for the [M+2H] 2+  and [M+3H] 3+  precursor ions of angiotensin II ( FIG. 10 ) and the [M+2H] 2+  precursor ion of pTK ( FIG. 11 ). In each case, fs-LID activation provides additional product ions compared to CID ( FIGS. 5 and 8 ), thereby providing increased confidence for assignment of the peptide sequence. 
         [0045]    The present fs-LID MS system and method achieves photodissociation of structurally important chemical bonds in large biomolecules. In  FIG. 14 , a comparison of expected results is presented between conventional CID and the present fs-LID on the isolated singly protonated precursor ion of angiotensin II (10 μM in methanol/water/acetic acid (50:50:1)). Fs-LID spectra is accumulated over 15 minutes using 200 ms irradiation periods, and reference CID spectra is collected at matching q-values. The CID spectrum ( FIG. 14A ) should provide ˜17 assignable peaks. In contrast, fs-LID of the [M+H] +  precursor ion ( FIG. 14B ) should provide more than 40 assignable peaks. The spectrum is particularly information-rich, especially when compared to the traditional CID spectrum to be collected under identical conditions, where fragmentation favored formation of the y 7  ion as well as ammonia loss from the singly protonated precursor. Notably, CID-MS 3  of the abundant fs-LID [M+H] 2+•  photoionization product expected to be observed at 523.1 m/z in  FIG. 14B  is found to give rise to many of the a and z ions observed in the MS 2  fs-LID spectrum ( FIG. 14C . Additionally, losses of p-quinomethide (−106 Da), COOH• (−45 Da), and [C 3 H 8 N 3 +H] +•  (−87 Da) are expected to be observed, consistent with the charge-remote fragmentation of peptide radicals previously described by (30) fs-LID MS 3  of the [M+H] 2+•  photoionization product should reveal that many of the low-mass b-type product ions in the MS 2  fs-LID spectrum are generated by further electronic excitation of the [M+H] 2+•  ion-radical in the strong laser field. 
         [0046]    Hence, fs-LID is capable of significantly increasing the number of sequence-relevant bond cleavage product ions, and that it is compatible with commercial ion-trap mass spectrometers. The additional flexibility provided by fs-LID coupled to ion trap mass spectrometry is demonstrated by obtaining MS spectra with either activation method (i.e., CID or LID). It is particularly worth noting that fs-LID should create a multiply charged radical molecular ion by removal of an electron, and that dissociation of this species gives access to additional valuable sequence information. Previously, the ability to acquire this information by photodissociation techniques has required the presence of a native chromophore or the introduction of a chromphore through chemical means. Thus, a significant advantage of fs-LID over previous photodissociation approaches is that no chemical treatment or the use of chromophores is required. 
         [0047]    In addition, fs-LID can also achieve photodissociation of modified peptides without losing valuable information about the specific location of the modification. CID-MS/MS of the singly protonated precursor ion of a model synthetic phosphopeptide GAILpTGAILK (pTK) (10 μM in methanol/water/acetic acid (50:50:1)) leads primarily to loss of 98 Da (H 3 PO 4 ), indicated in the expected spectrum by the Δ symbol ( FIG. 15A ). Most of the product ions have lost the phosphate group. Only the y 5 , b 9  and y 8  product ions preserve the phosphorylation. The reason why the phosphate groups (as well as many other protein modifications) are lost during conventional CID is that the activation process is ergodic, proceeding from weaker to stronger bonds. Given that many modifications have weaker bonds; these are lost early in the conventional CID process. In addition, the modifications may be prone to migration within the peptide, especially when the peptide gains energy through the CID process. This scrambling of information makes the correct assignment of protein modifications very challenging by CID.  FIG. 15B , however, presents expected result by fs-LID under identical ion-trap conditions as those used for CID. Here, exposure the intense field of the femtosecond laser cause extensive backbone cleavage in which the majority of the fragments (&gt;80%) retain the phosphate modification. More impressive is the expected MS 3  data in which the doubly charged radical ion generated by fs-LID is then subjected to CID. In this case, shown in  FIG. 15C , 100% of the fragments are expected to maintain the phosphate group. 
         [0048]    The present invention is more specifically employed to quantitatively evaluate the use of phase optimized fs-LID for protein sequence analysis. In other words, to initiate the optimization of phase-shaped laser pulses to promote diagnostically useful fragmentations such as those involving cleavage of selected bonds within peptide or protein ions. These cleavages may result in the formation of N-terminal b- and C-terminal y-type ions via cleavage of the peptide C—N amide bonds, or a-, c-, x- and z- type ions resulting from cleavage of the N—C or C—C bonds along the peptide backbone. It is especially desirable to obtain a complete series of these product ions because the mass difference between consecutive members of a series of such ions corresponds to the mass of an amino acid residue, thereby allowing the sequence or primary structure of the peptide or protein to be determined. 
         [0049]    The method of using this present system for such analysis is as follows. Peptides are introduced by infusion, or by on-line capillary RP—HPLC, directly coupled to a linear quadrupole ion trap mass spectrometer  101  (see  FIG. 19 ) (Thermo model LTQ, San Jose, Calif., USA) equipped with a nanospray ionization (nanoESI) source  103 . Quadrupole rods  105 , an entrance electrode  107  and an exit electrode  109  define a linear ion-trap  111  therein. A pair of mass spectrum detectors  113  are also provided. Individual precursor ions are isolated and subjected to CID or fs-LID. The current instrument configuration allows for selection of either CID or fs-LID at any stage of the analysis, thus providing great flexibility for experimental ion characterization. 
         [0050]    The laser is first optimized to deliver transform-limited pulses with 35 fs in duration, with 28 nm bandwidth centered around 800 nm to the 3D ion trap. Cancelation of phase distortions is achieved using the MIIPS software. The laser is operated at a 1 kHz repetition rate and the beam attenuated to 300 μJ/pulse (300 mW average power) and focused into the ion trap with a peak power of approximately 3×10 13  W/cm 2  at the center of the trap. These conditions should deliver good quality spectra after a single 300 ms activation window. Averaging of several such spectra should increase the reproducibility of peak heights. 
         [0051]    In a further embodiment, the present invention is more specifically employed to apply fs-LID to the improved identification and characterization of post-translational modifications in proteins from a biological source, starting with phosphorylation. The true value of the fs-LID methodology for accelerating human health research can be judged from its ability to generate useful information about important modified proteins derived from a biological source. The greatest challenges in PTM characterization are presented in the form of PTMs in large (&gt;2000 Da) tryptic peptides with multiple possible modification sites. These frequently yield conventional CID fragmentation that is inadequate to localize a PTM. 
         [0052]    Femtosecond laser induced ionization and dissociation leads to the formation of a large number of product ions, even in the absence of a native or chemically introduced chromophore. Analysis of the product ions reveals much more complete sequence coverage together with a much greater number of product ions that confirm the amino acid sequence and therefore increase the success rates when using an automated spectral analysis database. Furthermore, in contrast to the conventional method of collision induced dissociation, which often leads to extensive phosphate group loss or phosphate group scrambling of phosphorylated peptides, fs-LID of the present system leads to minimal loss of the phosphate group. Furthermore, fs-LID and CID can be used in the same instrument and are mutually compatible, thereby allowing MS 3  experiments in any combination, e.g., isolation:fs-LID:isolation:CID, and isolation:fs-LID:isolation:fs-LID, isolation:CI D:isolation:fs-LI D. The fs-LID technology provides the potential to deposit energy into selected ions in an efficient and controlled fashion independent of ion charge environment. This approach provides access to different kinds of ions that can undergo fragmentation through channels not available through conventional ion activation technologies. Such a technology offers substantial expansion of the ability to measure key regulatory events in a wide range of biological processes. 
         [0053]    For example, the present laser system allows for the quantitative evaluation of the use of phase optimized fs-LID for protein sequence analysis, and the application of fs-LID to improve identification and characterization of post-translational modifications in proteins, starting with phosphorylation. 
         [0054]    The results provide a quantitative assessment as to the usefulness of fs-LID in biological mass spectrometry. This establishes conditions for the effective use of ultrashort pulses in mass spectrometry for improved proteomic analysis. The advantages of the present system are realized when comparing the CID MS/MS spectra of modified peptides, for example histone proteins which are subject to modifications, such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, and ADP ribosylation, some of which are known to play important roles in the regulation of chromatin structure and function, with those obtained by fs-LID. fs-LID has the ability to achieve unambiguous assignment of the modification sites within these peptides. The present system is used to independently determine the modification sites and the advantages of the present system are greatest for proteins containing multiple modification sites. Regulation proteins are known to contain more than 20 post-translation modifications. The present system, therefore, results in a powerful new mass spectrometry instrument that achieves increased sequence coverage, and unambiguous assignment of sites and identities of post-translational modifications, while avoiding time-consuming chemical processes such as the addition of a chromophore, or derivatization. The speed with which fragmentation occurs with the present system minimizes possible position scrambling and loss of the modifications of interest, resulting in greatly improved assignment. 
         [0055]    Ultrashort laser pulses, less than 1 ps, preferably less than 60 fs and more preferably less than 30 fs, having a preferred wavelength greater than 700 nm and a preferred peak intensity greater than 10 12  W/cm 2 , can deposit energy by multiphoton transitions which are not commonly observed with conventional laser pulses and can induce field ionization. By modulating the spectral phase of ultrashort pulses, it is possible to control the amount of energy that is deposited and the subsequent fragmentation of the target ion. Essentially, the yield of each fragment ion produced is affected by the shaped laser pulses; this process non-ergodically focuses the available energy on specific chemical bonds in a timescale much faster than the rate of intramolecular energy randomization. The present system focuses a shaped femtosecond laser pulse on a designated precursor ion, and provides photodissociation fragmentation data which is used to elucidate the structure of the target ion. Pulse shaping is used to control the extent of photodissociation and to direct photodissociation to specific molecular motifs. These femtosecond laser pulses provide an attractive alternative to conventional CID methods that provide some fragmentation information but without the degree of user-directed control that will be possible with the present system. More notably, the femtosecond laser pulses of the present system avoid the thermalization process that accompanies conventional CID which leads to cleavage of the weakest bonds, and may lead to molecular scrambling in the activated species. Thus, the present system gives an active and selective energy source which providing the analyst with a ‘spectroscopic scalpel’ to generate structurally diagnostic fragment ions never before available for the elucidation of protein structure. 
         [0056]    fs-LID of the present system further ionizes a precursor and/or product specimen by removing at least one electron of the specimen. This is possible due to the preferred less than 1 ps duration and greater than 700 nm wavelength of the laser pulses. This desirable electron removal is not achieved by conventional CID or conventional use of laser pulses of greater durations and/or shorter wavelengths. 
         [0057]    A major barrier to the utilization of traditional femtosecond laser pulses was the expense and typically they needed optimization by a laser expert in order to yield reproducible results. The preferred use of MIIPS in the present system overcomes conventional difficulties in measuring phase distortions and correcting for them. MIIPS is an adaptive procedure that measures and automatically eliminates spectral phase distortions in seconds. Briefly, the MIIPS method is based upon monitoring characteristic changes occurring in the spectrum of a nonlinear process, such as second harmonic generation (“SHG”), when the phase of the input pulse is altered. In MIIPS, a pulse shaper with a programmable spatial light modulator (“SLM”) is used to introduce a reference phase function ƒ(λ), and the algorithm searches for wavelengths that satisfy the equation φ″(λ)−ƒ″(λ)=0, where φ(λ) is the unknown spectral phase of the laser pulse at the focal plane. Finding the values that satisfy the equation above is as simple as scanning a range of quadratic reference phase functions (amount of linear chirp) and collecting an SHG spectrum for each such phase. From the resulting spectra obtained as a function of the reference phase, the function φ″(λ) can be directly obtained. After its double integration, the original spectral phase φ(λ) is known, and a compensation phase (negative of the measured phase) is introduced to obtain TL pulses at the sample. The procedure is fully automated and takes less than a minute. Note that since the second derivative of the phase is measured and corrected for all wavelengths within the pulse spectrum rather than at a single (central) wavelength, MIIPS automatically accounts for all higher orders of dispersion. The pulse shaper that performs MIIPS is preferably placed between the oscillator and the regenerative laser amplifier, which allows for obtaining shaped pulses without loss of laser intensity. By placing the MIIPS detector near the mass spectrometer and using a window that is similar to the one at the laser input port, the system is able to compensate for phase distortions introduced by the oscillator, amplifier and even the air as the ultrashort pulses make their way to the MS system. This MIIPS technology ensures reproducible MS results. 
         [0058]    With the present system, controlled fragmentation is achieved when using binary phase shaping of femtosecond pulses, where each pixel in the pulse shaper receives a value of 0 or π. The methodology is called binary phase shaping mass spectrometry (“BP-MS”). The binary phases are identified as BP#, where the number corresponds to the decimal value of the binary code used to generate the phase. For example, the phase function 0101101101 corresponds to BP365 and 0111111100 to BP1020 (1 corresponds to retardation by π for that pixel), as shown in  FIG. 12 . An advantage to the use of binary-phase shaping instead of arbitrary phase shaping is that it speeds the search for specific pulses that yield the desired bond cleavage by orders of magnitude. Expected results of a binary phase search are mapped as shown in  FIG. 12 , where the diagram represents the experimentally recorded search space for the binary phase control of pyridine ionization versus loss of HCN under strong field excitation. Notice that the fitness, calculated as the ratio of peaks a (m/z 52) and b (m/z 79) can be controlled from 0.87 to 2.57. The mass spectrum for two specific binary phases is shown below with their associated binary phase. The search map shows inversion symmetry; this is because addition of it to a phase function gives an equivalent phase function. Transform limited pulses, the bottom left and the upper right corners, lead to small a/b ratios where less fragmentation is observed. There may be more than one optimum solution, for example, methods for selective multiphoton excitation and for selective impulsive stimulated Raman scattering. These approaches can be used to deliver energy selectively to the molecule. 
         [0059]    Typically, sets of experiments are programmed on the computer controller which records mass spectrometry data for each of the differently shaped laser pulses. Once the entire data set is obtained (typically about 20 minutes) the data is analyzed by plotting a particular desired outcome (for example the ratio between two fragmentation pathways) as a function of the binary phase number. As can be seen in the example given in  FIG. 12 , there is a large range of variation within the different shaped pulses. After testing, specific shaped pulses or pulse-sequences, which cause the desired fragmentation, are employed thereafter. Because the system can evaluate hundreds of shaped pulses per minute, this process is fast and efficient. Furthermore, once those pulse sequences are identified, they do not need to be searched again, they can be used as one would use a chemical reagent. 
         [0060]    In mass spectrometry, multidimensional analysis is helpful for molecular identification because there are a number of chemical species that are very similar and difficult to distinguish. Molecular isomers are species with the same chemical formula but different structure. With large biomolecules this occurs often. The ability to induce structure-sensitive photodissociation greatly simplifies the task of identifying molecular isomers. For example, ortho and para-xylene have identical electron-impact mass spectra. Binary phase shaping with MS is used to identify ortho- and para-xylene, something that electron impact MS cannot. 
         [0061]    In  FIG. 13 , the left panel shows the expected electron impact mass spectrum obtained from both compounds. The right panels show the ratio expected to be obtained between the molecular ion and the tropylium ion are clearly different for BPO and much more different for BP858. The ratios obtained can be used to identify these two isomers. In fact, these pulse sequences are used for the fast (0.1 sec) and reliable quantification of mixtures of both of these compounds. Shaped laser pulses are used to identify among a large number of isomeric pairs (positional, geometric and even some stereoisomers). This capability is applied to mass selected peptide ions as described below. Because the energetic requirement for ionization (equivalent to six laser photons) has already been overcome when the laser interacts with ionized peptides, it is expected to gain better control over fragmentation. The greater ability to cleave chemical bonds and to determine structural information such as type of isomeric species will assist proteomic and metabolomic analysis. Unlike the use of genomic biomarkers, characterization of protein—protein signaling by identification of phosphorylation states of proteins relates directly to cellular responses during disease progression or drug treatment. Thus, the systematic identification and characterization of phosphoproteins, including determination of the specific site(s) of phosphorylation within a protein of interest and quantitative analysis of temporal changes in phosphorylation status, are helpful to the development of a more complete understanding of the role of these modifications in the onset and progression of disease, and for the development of therapeutic strategies for their treatment. The mass spectrometry-based technique improves protein identification and characterization of phosphorylation sites compared to existing technologies. 
         [0062]    For all of the embodiments and uses disclosed herein, the ion-trap mass spectrometer preferably includes a 3D ion trap, but can alternately include a linear ion-trap, an ICR ion-trap, or an electrostatic Orbitrap, although focusing of the laser beam pulse on the ions may need to be adjusted accordingly. Moreover, quadruple or time-of-flight (“TOF”) analyzers may also be used depending on the specific application. It is additionally envisioned that the present system can employ various combinations of pulse characteristics (e.g., shapes, iterations, durations, etc.) and/or other steps including CID, fs pulses, and/or less preferably electron impact methods, to the targeted specimen being analyzed. Such combinations are automatically operated by a software routine stored in memory in the programmable computer, which are responsive to initially sensed iterative results and/or predetermined calculations. 
         [0063]    Referring to  FIG. 20 , software instructions are programmably stored in memory, such as RAM or on a disc, of a computer  69  (see  FIGS. 1 and 19 ) which runs the software in a microprocessor. The computer includes input devices, such as a keyboard and detector(s)  67  and  113  (see  FIGS. 1 and 19 , respectively). Moreover, output devices, such as a display screen and printer, are attached to the computer for visually showing mass spectrum information, laser pulse shapes, duration and other characteristics, and/or operator prompts. The software provides a data-dependent acquisition of MS/MS and MS n . 
         [0064]    The software can be run in a manual operator prompting mode, a fully automated mode, or combinations thereof. In the fully manual mode, the operator must analyze the MS information and physically enter one or more commands and/or settings to begin the next process step. In the fully automated mode, however, the software automatically analyzes the MS information obtained from fs-LID, such as by comparing it to target desired values or ranges, and then determines if a desired result has been obtained. If not, the software automatically isolates the precursor ions of interest of those previously ionized or in a new ion-trap fill from the same source specimen by causing frequency synthesizer  65  (see  FIGS. 1 and 19 ) to expel the undesired ions and retain the desired precursor ions through computer controlled energization of the power supply  63 . The software again automatically analyzes and determines if the desired mass spectrum results are obtained. If not, the software automatically isolates the product ions of interest, optionally decides which supplemental fragmentation process to run (e.g., CID, fs-LID again, SID, IRMPD, UVPD, ECD ETD, PSD, EID, EED, EDD, or MAD). An optional set of instructions allow for manually or automatically controlled modification of a laser pulse characteristic and/or operation of the shutter. For example, the pulse shape or duration can be varied between pulses or series of pulses. 
         [0065]    It is noteworthy that ionizing an ionized specimen by removing at a least one electron can create multiply positively charged, singly negatively charged or multiply negatively charged ions. It is also worth noting that fs-LID fragmentation and modification of a sample can optionally be facilitated and directed by the addition of high atomic number metal counter-ions. Furthermore, when the term “sample,” “specimen” or “same specimen” is used, it includes both situations, where a specific precursor ion is transformed into a product ion which is itself further ionized/fragmented, or where the ion-trap is refilled or reloaded with new portions of the same specimen batch between each ionization/fragmentation process, including multiple separated processes thereon. 
         [0066]      FIGS. 16-18  show various configurations of a three-dimensional ion-trap  161 ,  163 , and  165 , respectively, for a mass spectrometer of the present system. A laser pulse  43  is focused in the ion-trap by lens  85  and directed to an ion packet or cloud  167 .  FIG. 16  illustrates a single pass cavity in the mass spectrometer, where the pulse is transmitted in a single direction from lens  85 , to ion packet  167  then to a mirror whereafter it is reflected away from ion-trap  161 .  FIG. 17  shows a double pass cavity in the mass spectrometer. In this configuration, a concave mirror  169  reflects pulse  43  back into ion-trap  163  to act upon ion packet  167  a second time. 
         [0067]    Referring to  FIG. 18 , a multipass cavity in the mass spectrometer employs a pair of concave mirrors  171  and  173 , a flat main mirror  175 , and a flat and smaller entry mirror  177 , and a flat smaller exist mirror  179 . The multipass mirror  171 ,  173 , and  175  generally surround at least three sides of ion packet  167 . The multipass cavity construction causes each laser pulse  43  to act up the ion packet multiple times, in this example, at least six times and more preferably, nine times in order to irradiate at least a majority of the ion cloud. This advantageously provides more efficient and quicker ionization and/or fragmentation, which further activates the ionized specimen faster than intramolecular energy redistribution can occur. Furthermore, a single pulse (of a series of pulses) acts multiple times on the ion cloud, where each reflection of the pulse therethrough has the identical pulse characteristics. This is also expected to provide stronger and more easily identifiable mass spectrum information since more abundant data will be generated with greater sensitivity. The multipass cavity configuration is ideally suited for ultrafast and high intensity pulses, such as those of less than 1 ps, with an intensity greater than 10 12  W/cm 2  and a wavelength greater than 700 nm, and especially with a 3D or linear ion-trap mass spectrometer. 
         [0068]    Another use is for identifying PTM in pharmaceuticals, finding disease markers in molecules, and for metabolic analysis. Further uses include identifying proteins, DNA and RNA for forensics, and to provide a disease prognosis and appropriate corresponding therapies. The present method can alternately be used for disease diagnosis, monitoring disease progression, detecting the presence of a drug, determining stress-related modification and determining predisposition to a disease, through the present fs-LID determination, detection and/or identification of PTMs and metabolites. Another use of the present method is for the study of metabolites. The present method leads to the cleavage of strong bonds that are not usually cleaved by CID. For example, it leads to cross-ring fragmentation in carbohydrates. These types of non-conventional fragmentation patterns are very helpful in metabolomic analysis because they provide additional information that can be used to elucidate the identity and structure of the metabolites. Metabolites can be significant markers for disease, and therefore, monitoring can aid diagnosis and the determination of disease progression. Similarly, pharmaceuticals are metabolized and the resulting metabolites can lead to undesired side effects. Therefore, the present system represents an important new tool for metabolomic analysis for a broad range of small molecules including but not limited to carbohydrates, lipids, steroids, ketones, glycols. 
         [0069]    Various embodiments of the present invention have been disclosed but modifications may be made. For example, the present system can optionally be used without MIIPS although many of the advantages may not be realized. Furthermore, a pulse shaper located downstream of the laser amplifier and oscillator may alternately be employed. Additional, fewer or differently placed reflectors, such as mirrors, can be used. Other methods for determining mass to charge such as ion mobility, time-of-flight, reflection, and other types of magnetic or electric lenses and traps, whether they are large or miniature, may be used instead or in addition to those disclosed. Similarly, sample preparation, solvents and their concentrations are typically adjusted to yield a stable ion source. While various optics and equipment types have been disclosed, other devices may alternately be employed as long as the disclosed function is achieved. It is intended by the following Claims to cover these and any other departures from the disclosed embodiments which fall within the true spirit of this invention.