REVIEW

Wolfgang Schrader Æ Heinz-Werner Klein

Liquid chromatography/Fourier transform ion cyclotronresonance mass spectrometry (LC-FTICR MS):an early overview

Received: 16 December 2003 / Revised: 19 April 2004 / Accepted: 10 May 2004 / Published online: 23 June 2004

� Springer-Verlag 2004

Abstract Fourier-transform ion cyclotron resonancemass spectrometry has developed into one of the mostpowerful analytical techniques. This unique techniqueenables acquisition of high-resolution mass spectra withhigh accuracy, which in turn enables determination ofthe elemental composition of the analyzed compounds.Coupling with liquid chromatography affords a separa-tion technique with a high-resolution ‘‘detector’’ whichcan be used to investigate very complex matrices. In thisreview some important instrumental developments aredescribed and applications are presented; these show theadvantages and disadvantages of this combination.

Keywords Liquid chromatography Æ Fourier-transformion cyclotron resonance mass spectrometry ÆLC–FTICR Æ Proteomics Æ High-resolution massspectrometry

Abbreviations CAD: Collision-activated dissociation ÆCZE: Capillary zone electrophoresis Æ ECD: Electron-capture dissociation Æ FT: Fourier-transform Æ MS:Mass spectrometry Æ ICR: Ion cyclotron resonance ÆIRMPD: Infrared multi-photon dissociation Æ LC:Liquid chromatography Æ LSIMS: Liquid secondary-ionmass spectrometry Æ SORI: Sustained off-resonanceirradiation

Introduction

Ion cyclotron resonance (ICR) spectrometry is a tech-nique that has been used since the 1960s for studies of

gas-phase ions, in particular ion–molecule reactions [1,2]. One feature that connected ICR with such techniquesas nuclear magnetic resonance spectroscopy (NMR) andinfrared spectroscopy (IR) is that a broad frequencybandwidth had to be slowly scanned. As was the casewith the other techniques, ICR became a faster andmore efficient analytical technique with the developmentof Fourier data reduction that was introduced in 1974 byComisarov and Marshall [3, 4]. The addition of Fouriertransformation (FT) makes Fourier-transform ioncyclotron resonance (FTICR) a faster scanningtechnique with broadband detection and better signal-to-noise ratio. Since the middle of the 1990s, in partbecause of developments in instrumentation andmicroelectronics, FTICR mass spectrometers have beentransformed from expensive, extravagant, and sophisti-cated research instruments to efficient tools for highlycomplex analyses [5].

The coupling of liquid chromatography (LC) to massspectrometry (MS) is nowadays well established inroutine analysis. However, one question that has to beanswered is, why would it be necessary to use anexpensive instrument like an FTICR as ‘‘detector’’ forchromatography, when cheaper alternatives are avail-able? What are the needs for a mass selective detectoranyway?

One can argue, that the needs depend on the problem.Sometimes, though, the problems become more com-plex, and higher resolution or accuracy is needed eventhough chromatographic separation has been applied.Also, if complex spectra are compared with data from adatabase, the better the quality of the spectra the betterthe search results. Another feature that is appealing isthe structural information that can be obtained fromfragmentation experiments, generally termed as MS–MSor MSn.

Since the introduction of electrospray ionization(ESI) [6, 7] it has become the interface of choice for thecoupling of LC with MS. Although matrix-assisted laserdesorption ionization in combination with a time-of-flight (MALDI-TOF) instrument enables measurement

This contribution is dedicated to Professor Dr M.T. Reetz on theoccasion of his 60th birthday.

W. Schrader (&) Æ H.-W. KleinMax-Planck-Institut fur Kohlenforschung,Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, GermanyTel.: +49-208-3062271Fax: +49-208-3062982E-mail: wschrader@mpi-muelheim.mpg.de

Anal Bioanal Chem (2004) 379: 1013–1024DOI 10.1007/s00216-004-2675-1

of accurate mass data, it is not suited for coupling to LCand for MS–MS measurements. ESI-TOF instrumentsenable easy combination with LC, but still do not enablereal MS–MS. Triple-quadrupole and ion-trap (IT) MSare equally well suited for LC coupling and enable MS–MS experiments, but although mass accuracy and massresolution have improved in the last generation ofinstruments, they are still neither high-resolution norhigh-accuracy instruments. In the last few years, thehybrid instrument resulting from combination of aquadrupole and a TOF analyzer has had much success,because it enabled coupling with LC and MS–MScapabilities, and mass resolution above 10,000 combinedwith mass accuracy better than 10 ppm [8].

Nonetheless, for complex mixtures these capabilitiesare sometimes not enough. Especially in biological orbiochemical-oriented research, for example proteomicsor genomics, extracts from protein digests can be verycomplex. The complexity of such samples is increased bythe appearance of multiple higher-charged ions whichsometimes can only be resolved with very high resolu-tion.

FTICR instruments, on the other hand, afford highresolution (�150,000) and accurate (�2 ppm) massspectra on a routine basis. The reason for this perfor-mance lies in the unique technique used for mass anal-ysis. Whereas in other mass spectrometers ions arefiltered in a magnetic or electric field or selected by flighttime, in FTICR instruments ions are detected by theirresonance frequency; this can be measured accuratelywith today’s electronics and, therefore, accurate resultscan be obtained.

In the last few years, the number of papers publishedon LC–FTICR has increased because of the highernumber of instruments that have been sold to labora-tories worldwide. So it seems an appropriate time tointroduce an overview of the possibilities that arise fromcoupling of these techniques. Whereas Pinto et al. [9]recently reviewed the overall capabilities of FTICR, thisreport will focus on instrumental developments thathave made LC–FTICR possible and on some biochem-ical, combinatorial, and environmental applications.Fragmentation techniques that have been found usefulin all kinds of application are also reported. Nonethe-less, the emphasis will be placed more on the ‘‘detector’’and the applications rather than on the development andimprovements of the separation.

Principle of ion cyclotron motion

The general principle of FTICR is described in detail intwo reviews by Marshall et al. [10, 11]. To emphasize thepossibilities of the technique a brief introduction is givenhere. The illustration in Fig. 1 shows the basic set-up ofa cyclotron cell, which consists of three pairs of adjacentelectrodes (plates) that are assembled like a cube or, inother cell designs, like a cylinder. The front and endplate work as trapping plates to trap the ions inside thecyclotron cell. The two excitation plates are connectedwith a radio-frequency (RF) transmitter to ‘‘excite’’ theions and the detection plates register the induced mirrorcurrent of the ions for detection.

In general, ICR spectrometry is based on the princi-ple of cyclotron motion in a uniform magnetic field. Ionsare detected in a cyclotron cell, which is located inside asuper-conducting magnet with a fixed field strength(currently magnets for commercial instruments areavailable with 4.7, 7, and 9.4 T which corresponds to

-

magneticfield B0

fastFourier

Transformation

frequencyspectrum

mass spectrum

rf excitation

detection plate

Fig. 1 Schematic diagram of the ICR cell, consisting of twoopposite excitation plates (right and left), two detection plates (topand bottom) and two trapping plates (front and back). Ions enterthe cell through the front trapping plate

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proton Larmor frequencies of 200, 300 and 400 MHz,respectively). The first 12 T magnet has recently beendelivered.

Ions arriving inside the cyclotron cell are forced intoan orbit by the uniform magnetic field. Two differentcases are possible:

– for ions traveling in the same direction as the magneticfield, the direction is not affected; and

– ions not moving in the same direction as the magneticfield are forced into a circular orbit.

This force can be expressed as:

FL ¼ qðv� BÞ ð1Þ

where F is the force, q the charge of the ion, v thevelocity of the ion, and B the magnetic strength.

The velocity and magnetic field vectors are perpen-dicular to each other, causing the cross product to beperpendicular to v and B with its magnitude being theproduct qvB, with positive and negative ions moving inopposite directions.

Two competing forces can explain the cyclotronmotion, in which the outward directed centrifugal forceFc is compensated by the Lorentz force FL. On a stabletrajectory the two forces are equal, so we get for FL=Fc:

qvB ¼ mv2

rð2Þ

which results in:

vr¼ q

Bm

ð3Þ

From Eq. 4, the cyclotron frequency x can be expressedas:

x ¼ qBm

ð4Þ

where x is the cyclotron frequency, m the mass of theion, and r the radius of the trajectory.

It has to be noted that the final equation does notinclude the velocity of the ion, thus ions of one masshave the same cyclotron frequency, irrespective of theirvelocity. Equation 4 gives the results in radiation persecond and has to be converted to frequency in Hertz bydividing by 2p.

The ions are thus ‘‘stored’’ inside the cyclotron cell. Ifan RF pulse is sent to the cell the ions will gain energywhen the RF field is equal to the cyclotron (resonance)frequency of the ion; they therefore move into a largerorbit. The RF frequency is transmitted by the excitationplates of the cyclotron cell. Most instruments use an RFsweep that covers the range between several kilohertzand the lower megahertz range to accelerate the ionstrapped inside the cell. This sweep pulse is called a chirp[10].

After this energy uptake the ions circulate the cell inthe higher orbit, thus getting closer to the detectionplates and inducing a stronger electrical ‘‘mirror cur-

rent’’; this signal is subsequently amplified by the elec-tronics. The circular rotation produces a signal from allions at approximately the same orbit, but at differentfrequencies for each ion which, with time, loses energyand drops back to a lower orbit. The transient signalfrom all the ions is then digitized and processed with afast FT algorithm, resulting in a ‘‘traditional’’ massspectrum with ion abundance versus mass-to-chargeratio (m/z).

Coupling liquid chromatography to FTICR

The biggest difficulty of coupling LC to FTICR is thebalance between the time available while a separatedcompound is leaving the chromatography column andthe scanning time required for a sensitive detection. Fordetection in an ICR cell a certain number of ions mustbe collected. Marshall et al. [10] calculated that undertypical conditions, approximately 187 ions would berequired for a signal-to-noise ratio of 3:1.

For a sensitive and accurate ion detection the ionshave to be trapped inside of the cell. In addition to themagnetron and cyclotron motions in the cell electricfields play a significant role, i.e. in the case of oscillationbetween the plates. The influence of the ion trapping canbe critical to the performance of the ion detection.

One method used to collect the ions inside the cell is‘‘gated trapping’’ [12], where the front end electrode ofthe ICR cell is lowered to ground. While the ions aremoving through the FTICR cell the electrode is rapidlyraised to trap them, followed by excitation and detec-tion. Disadvantages of the combination with a contin-uous source is a low duty cycle.

Another, similar, method is off-axis deflection trap-ping [13], where the ions are deflected while entering thecell. The deflection enables a longer residence time,which is extended from 1 ms to tens of ms [14]; this leadsto an increase in the number of ions collected. Some ofthe disadvantages are increased radial diffusion and side-effects on excitation and detection caused by a non-centered ion cloud.

The gas-assisted trapping method [15] in which ionsare decelerated and trapped by collisions with neutralbackground molecules while passing through the trap isvery effective. An improved version of this method usedpulsed gas-assisted trapping [16] for collection of theions. For both methods a background pressure of atleast 10)5 torr was necessary to trap ions effectively. Thedifficulty, however, is that the pressure for detection inthe FTICR cell has to be lower than 10)8 torr. Thisapproach is, therefore, unsuitable for LC coupling forcomplex systems because the acquisition rate of theFTICR is much slower than the LC elution times.

One means of solving this problem has been the useof an external octapole, located after the ion source, forion accumulation and ion cooling. Because of the higherpressure in this region, ion cooling is easier and does notneed an additional gas load. The concept, which has

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previously been demonstrated on IT MS and on triple-quadrupole instruments [17–19], was developed for anFTICR by Senko et al. [14]. In comparison with theconventional techniques of cooling the ions directlyinside the Penning trap, external storage enables fasteracquisition and better spectrum quality because thevacuum in the ICR cell is not affected by the long ion-accumulation times, where the high vacuum cannot bemaintained. This development enabled sensitive cou-pling between LC and FTICR.

It was recently reported [20], however, that accumu-lation in multipoles can lead to discrimination of smallerions m/z. This is caused by space charge repulsion andion separation because of m/z-dependent balance of theeffective potential force. Another effect is the spacecharge-induced instability against lower m/z ions [20].To avoid these ion discrimination effects, a loweramplitude dipolar excitation has been superimposed onthe main RF-field or increased pressure in the multipolewas applied through a gas pulse during accumulationperiods. Additional work has been performed on usingimproved accumulation procedures in the form ofautomated gain control (AGC) [21] where the amount ofions accumulated is determined through pre-scan data.This enables to avoid overfilling of the external trap andoptimization of accumulation parameters. A similarconcept has already been developed for 3D IT [22].

Home-built instruments

Some of the early work on LC–FTICR MS was per-formed using home-built instruments. One of the firstinstruments designed for coupling to LC came from theAmerican Cyanamid Corporation. Their need for sen-sitive, accurate, reproducible, and highly resolved massspectrometric data made it necessary to build a machinethat was not commercially available at that time [23].The authors constructed an instrument with an externalion source that was capable of being connected to LCand GC. LC coupling was realized with the use ofa Vestec thermospray [24] and an liquid secondary-ionmass spectrometry (LSIMS) source [25] using a Cs-gunoperating at 9 kV. The instrument was equipped with a7-T superconducting magnet.

For LC–FTICR applications the Marshall groupoptimized two instruments with 9.4 and 7 T magnets[26–28]. First results have been reported using the 9.4 Tinstrument in combination with a home-built micro-ESIsource constructed as a Chait-style interface [29].

The performance of FTICR instruments depend lar-gely on the ultra high vacuum in the cyclotron cell. Toensure this is maintained with the LC it is advantageousto reduce the gas load that can be associated with astandard LC–ESI-MS combination. Therefore, Emmettet al. [26] designed a special micro-ESI needle for ESIwith an inner diameter of 25 lm. This system enablesuse of nano-LC conditions for FTICR, thus keeping thegas load to a minimum.

Although the first instrument [26] was capable ofobtaining results from low concentrations at the femto-mole level, improvement of an instrument with a 7-Tmagnet resulted in greater sensitivity. By reducing theoctapole length from 60 to 15 cm, increasing the diameterof the ESI emitter to 100 lm, and using new RF-poten-tials the sensitivity was improved to the attomole level[27].

Martin et al. [30] constructed a spectrometer that usesexternal ion accumulation and two RF-quadrupoles astransfer elements in combination with an open Penningtrap. One attractive feature of this set-up is a self-mademicrocapillary HPLC column with integrated emittertip. This combines the column and the ESI emitter, thusshortening the transfer distance after separation andreducing the peak broadening that can arise from longtransfer lines and capillary connectors.

For the column a capillary of 30 cm length (360 lmOD, 50 lm ID) is used. About 1 cm of polyimide isremoved from the end of the capillary and a bottleneckrestriction is inserted by use of a laser puller. Afterwardsthe column is flushed with a slurry containing Poros 10R2 packing material to insert a plug that keeps theC18 reversed-phase material (5 lm) inside the column.By using the laser puller emitter tips are formed with5 lm diameter for MS and 1 lm for MS–MS measure-ments, to correspond to the ‘‘high-flow’’ and ‘‘low-flow’’modes used by the authors in these experiments.

The field of proteome research has grown rapidly inthe last few years, and MS-based methods have played abig role in the advances that have been made in this field.Protein analysis, though, can be complex. One successfulapproach is the use of 2D polyacrylamide gel electro-phoresis (2D-PAGE), which can be a powerful tech-nique in combination with MS [31, 32]. The problemsof protein analysis with 2D gel electrophoresis are thatcertain proteins can be lost or suppressed on the gel.

To provide a new tool for protein analysis that was notbased on a 2D-PAGE Shen et al. [33, 34] reported thecombination of reversed-phase LC with a 3.5-T FTICR[33] and the use of amultiple capillaryLC-system [34]withan 11.4-T instrument. This group also worked in collab-oration with the Ewing group [35, 36] on the coupling ofcapillary zone electrophoresis (CZE) to an FTICR.

The 3.5-T system was equipped with an ESI interfaceusing a heated metal capillary inlet and an electrody-namic funnel assembly [37]. In-house capillary columns(150 lm ID · 360 lm OD) were packed with bondedpacking material.

The 11.4-T LC–FTICR system consists of severaldual-capillary-column devices and operates at a pressureof approximately 68.95 MPa (10,000 psig). This systemimplements a passive feedback valve arrangement thatenables switching of mobile phase flows. This set-up usesseveral dual capillary columns, which are connected inseries to eliminate time delays and thus reduce analysistimes. The multi-column approach enables washing andequilibration of one column while the next is used forseparation.

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This FTICR utilized a linear quadrupole IT as asegment of the ion guide that enables use of the transferdevice to convert the ESI source into a pulsed sourceand to enhance the dynamic range, which in the end stillrequires 5.7 s per spectrum. For enhancement of spec-trum quality a small amount of nitrogen gas was intro-duced into the ICR cell to reduce translational energies[34].

Commercial FTICR instruments

Several FTICR instruments are currently availablecommercially. The machine with probably the longesttenure in LC–MS is the Bruker APEX instrument, cur-rently delivered in its fourth version. In the last few yearsseveral papers have been published on LC–FTICR usingthis machine with magnets of 7 and 9.4 T.

Most magnets nowadays are available with activeshielding. For direct coupling with LC an ESI source ora nano-spray source are available. Figure 2 shows aschematic diagram of the Bruker APEX III in an LCset-up with an LC Packings nano-LC system using anano-spray source [38]. Ion accumulation occurs afterionization in a hexapole from where ion packets aretransmitted through several cylindrical or half-cylindri-cal electrostatic lenses to the cyclotron cell.

A new instrument is the Thermo LTQ FTICR MSSystem (Fig. 3) [39] that combines a linear IT MS with

an FTICR analyzer. The concept is to use the IT as adevice to separate detection in the Penning trap from allother tasks. The IT can therefore be used as a massanalyzer, as a device for data-dependent ion accumula-tion and ion cooling, and for collision activation beforethe ion packets are transferred. For the transfer a seriesof differentially pumped RF-only multipole ion guidesare used.

Spectra can be obtained solely with the IT or addi-tionally after accumulation with the FTICR. This meansthat all functions that can normally be performed in anIT are available, with the added possibility of subse-quently running the ions through an FTICR. Thecombination is a concept which could enhance ionaccumulation and improve the duty cycle, thereforemaking it well suited for LC analysis.

A third FTICR is from Ionspec. Although someremarkable results obtained by use of this machine havebeen reported (see, for example, the work of Muddiman[40] or Zubarev [41]) applications of direct LC couplingare not available.

Bruker has recently also introduced the APEX-Q inwhich a Q-q front-end device is combined with anFTICR to enable data-dependent analysis. The purposeis to remove MS–MS experiments from the Penningtrap, which is supposed to improve spectrum quality.

LC–FTICR applications

Biochemical applications

When using an FT mass spectrometer capable of massresolution above 100,000 and mass accuracy around1 ppm on a regular basis, the use of chromatography is

H20, 0.1 % HCOOH

90 % ACN,0.1 % HCOOH

Micropump

Split

FamosTM Autosampler (LC Packings): Injection 1.0 µl

UltimateTM

Nano-LC system (LC Packings)

PepMapTM nano column (LC Packings)

75 µm inner diameter15 cm length

3 µm particle size

Fused silica capillary (20 µm inner diameter)

Flow150 uLmin

Flow 200 nL/min

Distal coated needle (New Objective, ID tip 75/15 µm)

Union

7 T actively shielded magnet

capillary Ion guide ICR cell

pumps pumps

Needle holder of the Online nanospray source (Bruker)

Fig. 2 LC–FTICR combination using an LC Packings nano-LCwith an FTICR (Courtesy of Bruker Daltonics, Bremen, Germany)[38]. The system is equipped with an electrospray source including amultipole for ion accumulation and an ion transfer systemconsisting of electrostatic lenses

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sometimes not required. It costs too much time to de-velop and run a separation, while the same results can beobtained from the highly resolved accurate mass spectra.Use of a separation technique before FTICR is, there-fore, worthwhile only when the analytical problem isextremely complex or sample preparation time can bereduced. One field for which LC–FTICR is particularlywell suited is proteome research, and this is where mostapplications have been reported.

As mentioned above, the first applications of LC–FTICR were reported by Stockton et al. [23, 42] usingtheir home-built cyanamid FTICR instrument. Theypublished results relating to minor components ininsecticides and herbicides, obtained using thermosprayionization, and investigated the fragmentation of syn-thetic peptides using LSIMS–FTICR.

One early report of the use of a commercial FTICRcoupled to gradient reversed-phase LC came fromStacey et al. [43]. The Bruker group used a CMS 47Xwith external ESI source from Analytica of Branfordand demonstrated the separation of five standardpeptides.

Since these two groups demonstrated the usefulnessof LC–FTICR, improvement of the instruments hasmade their use easier and more reliable. Quenzer et al.[28] not only demonstrated the possibilities with regardto accuracy and mass resolution, but tried to reduce thedetection limit of FTICR analysis. They were able to

detect the peptide Arg8-vasotocin in water at 100 amolon column and detection of 300 amol on column inbiological fluid was still possible. While demonstratingthe ability to obtain results from low concentrations inbiological samples they emphasized the need for clean-up steps that are required to desalt the sample and thusenable this sensitive detection.

Shen et al. [34] demonstrated the power of LC–FTICR analysis by separating a global soluble yeasttryptic digest using packed LC capillaries. They inves-tigated the sample capacity for packed columns 100 cmlong (150 lm ID) packed with 3-lm C18 particles of120 A pore size. Studies of Saccharomyces cerevisiae(yeast) haploid strain S288C illustrated the capabilitiesof LC–FTICR. In Fig. 4 a 2D view and the corre-sponding total-ion chromatogram (TIC) from separa-tion of a tryptic digest of the yeast strain showsapproximately 110,000 detected components. The au-thors noted that this should not be the end of thecapacity of FTICR analysis and it should be possible toanalyze even more complex systems [34].

A study of advanced glycation end (AGE) productswas performed by Marotta et al. [44]. AGE products arekey products of the Maillard reaction between sugarsand amino acids which is important in food and livingsystems. This reaction seems, among many others, to beresponsible for some degree of protein cross-linking, andhence for production of toxic compounds. Glycation ofhuman serum albumin (HSA) resulted in approximately20 glycated peptides, for which structures were postu-lated as a result of accurate mass determinations byexamining the known sequence of HSA.

7 T Actively Shielded Superconducting Magnet

FTMS Data

Linear Ion Trap MS• MS, MS/MS and MS^n Analysis• AGC Control• Secondary Electron Multiplier Detector

FTICR MS• Ion Image Current Detector• Accurate Mass• High Resolution

60 m3/hr 300L/sec 400L/sec 210L/sec 210L/sec15 L/sec

Linear Ion Trap Data

Triple Ported Turbo Pump

Fig. 3 LC–FTICR combination of a linear IT MS with an FTICR(Courtesy of Thermo, Bremen, Germany) [39]. The system isequipped with an electrospray source; ion transfer from the iontrap to the FTICR is accomplished by using a quadrupole ion guide

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A contemplative view of protein analysis from a 2Dgel with LC–FTICR was published by Ihling et al. [45].Although they demonstrated successfully the analysis ofprotein spots, from the gel, of membrane extracts ofCOS-6 cells from green monkey (Cerecopithecus aethi-ops) kidney and identified several proteins, the authorswondered whether analysis of 2D gel proteins was theright application for an FTICR. Rapid identification of2D gels is also possible using a cheaper MS [31, 32],which is more cost effective, whereas the strength ofFTICR is in analysis of highly complex samples.

In contrast, Witt et al. [46] demonstrated some of theadvantages of LC–FTICR in fast isocratic separation ofa protein digest. The high quality of the data obtainedwith this set-up makes it easier and faster to searchpeptide libraries with high scores using very narrowmass tolerances.

A new LTQ FTICR system from Thermo has re-cently been introduced. The first instruments have beendelivered and the first results from LC–FTICR appli-cations have been reported [39]. Horning et al. [39]presented the analysis of complex crude extract fromhuman blood platelets with a resolution above 300,000;this is shown in Fig. 5.

Combinatorial chemistry

Combinatorial libraries from parallel reactions in solid-phase or condensed-phase organic chemistry [47, 48]enable rapid preparation of new compounds. This ap-proach is especially useful in the drug-discovery process,but it is also challenging in analytical chemistry becausehigh-throughput [49] methods have to be developed toaccompany the synthetic developments.

Schmid et al. [50] reported the analysis of compoundlibraries with up to 144 pyrazole carboxylic acids and sixsub-libraries with 24 compounds by use of FTICR andLC–FTICR. Although components of the six sub-libraries were identified by direct infusion experiments,the compounds of the larger library could not be fullyidentified. Problems with direct infusion can occur—theauthors reported signal suppression with complex sys-tems—with the result that four compounds could not bedetected from the large library. Another problem wasthat only 138 compounds of the large library wereexpected to be measured because of the presence of sixisobaric pairs. The coupling of micro-HPLC to FTICR,however, enabled detection of all the compounds of thelibrary.

The authors stated that best results were obtained bydirect infusion with internal calibration, although signalsuppression prevented detection of all 144 compounds.

Environmental analysis

A totally different kind of application is represented byour own work. The ‘‘Blue Haze’’ phenomenon on sunnydays above forests has been known since the early 1960s.This effect is believed to result from organic aerosolsformed from biogenic emissions [51]. The formation ofthese aerosols is of great scientific interest, becauseaerosols are believed to influence the regional climate.They absorb, reflect, and scatter incoming solar radia-tion, and serve as cloud condensation nuclei; reportsindicate that they are involved in multiphase atmo-spheric chemistry [52, 53]. The aerosols most probablyresult from reaction of biogenic hydrocarbons, mostlymono- and sesquiterpenes, with atmospheric oxidants,for example ozone, OH radicals, or NO3 radicals. Foridentification of reaction products and investigation offormation pathways we studied this problem with dif-ferent analytical techniques. Volatile reaction productswere investigated using GC-coupled techniques whereaslow-volatility products had to be analyzed by LC. Wetherefore used the combination of LC with IR, NMR,

Fig. 4 Right: TIC of a global soluble yeast tryptic digest recordedafter LC–FTICR analysis, using a gradient from A (H2O) to 75%B (10:90 H2O ACN), with both containing 0.2% acetic acid and0.1% TFA (v/v) over 189 min at 68.95 MPa (10,000 psig). Left:Portion of the LC–FTICR experiment in 2D display; approxi-mately 110,000 components detected [34]. (Reprinted with permis-sion. Copyright [2001] American Chemical Society)

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and MS for these studies on the reaction of the mostabundant monoterpene, a-pinene, with ozone [54, 55].

Because these studies were successful and gave someinsight into a very complex reaction with more than 200signals from different compounds detected, we realizedthe combination of LC with an FTICR as well. TheFTICR used was a Bruker APEX III (Bruker Daltonics,Bremen, Germany) equipped with a 7-T actively shiel-ded magnet and an Agilent electrospray source. This set-up enabled routine mass resolution of approximately50,000 with a mass accuracy of better than 2.5 ppm.

Although the basic reaction products are chemicallysimilar, LC separation enables slight separation, whichis really necessary for IR, NMR, and standard MSstudies. This is one example in which coupling to LC didnot reveal more information than could be obtainedwithout. Although LC set-up, column equilibration, andgradient separation take time, continuous infusion of thesample gave the same results much more quickly, how-ever. The overall results revealed the complexity of thereaction, in which up to five isobaric compounds appearat one nominal mass, with a difference of 36 mDa, whichis the difference between CH4 and an oxygen atom; thiscan be seen in the insert in Fig. 6.

A number of reaction products have already beenidentified, mostly compounds that contain a carboxylgroup [56–58]. In addition to the primarily formedreaction products, not only were dimers of the carbox-ylic acids detected [59], but also trimers and tetramers(Fig. 6). The results from high-accuracy mass analysisenable characterization of these signals and could verywell explain the formation of aerosols from biogenicemissions.

In this work direct introduction seems the best way ofanalyzing the complex reaction mixture, especially con-sidering that results from direct infusion were moreaccurate then those from LC.

Fragment analysis

Electrospray as a soft ionization method mostly givesinformation about the molecular or quasi-molecular ionof a compound, but little fragmentation.

There are, however, instances when additionalinformation about the molecule is needed, in particularfor identification of the molecule, of a specific sequenceor of modification of the structure. Fragments of themolecular ion can deliver information about the struc-ture or substructure of the precursor molecule. In classicMS fragmentation occurs via ionization with electronsthat produces a characteristic pattern with substantial

Sample: very complex crude extract from human blood platelets

Amount: unknown, but very low conc.Flow: 200 nl/minScan Cycle: 1 spectrum every 3.5 s

HCT116_A_030523101055 # 2189 RT: 72.26 AV:1 NL: 7.84E4FTMS + p ESI Full ms [ 200.00-2000.00]

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HCT116_A_030523101055 # 2189 RT: 72.26 AV:1 NL: 7.84E4T: FTMS + p ESI Full ms [ 200.00-2000.00]

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Fig. 5 LC–MS analysis of a crude blood extract. The spectrum ofthe peak at 72.26 min is displayed and increased in size (Courtesyof Thermo, Bremen, Germany)

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fragmentation; this enables thorough characterization ofthe molecule. For soft ionization techniques, e.g. ESI,chemical ionization (CI), atmospheric pressure chemicalionization (APCI), or atmospheric pressure photo-ioni-zation (APPI), this fragmentation must be induced.Mass spectrometers with multiple analyzers or spec-trometers implementing an ion-storage device can gen-erate fragmentation data from a specific ion.

Fragmentation can be initiated by different tech-niques. A common method is to increase the internalenergy of the molecule, which subsequently results infragmentation. The most common way to create frag-mentation by collision-activated dissociation (CAD) isto cause collisions with neutral gas molecules. ForFTICR a technique called sustained off-resonance irra-diation (SORI) has been introduced [60].

Previously, collisional activation was achieved byapplying a short electric pulse (<500 ls) to the ions tocreate inelastic collisions. In SORI the molecules areexcited by a sustained (‡500 ms) electric field pulse.

Exciting the molecules with a long-duration off-reso-nance pulse leads to multiple collisions, resulting inhigher fragment intensities. In SORI the precursor ion isisolated first before being subjected to collisions insidethe Penning trap. The disadvantage for SORI is the highpressure inside the Penning trap, which reduces theperformance of the instrument. Other methods havetherefore been developed for fragmentation.

Infrared multi-photon dissociation (IRMPD)

An elegant way of causing fragmentation is to increasethe internal energy with an infrared laser, which is doneusing the IRMPD method. The use of IRMPD to vib-rationally excite ions has been known for some years[61]. IRMPD of ions in an FTICR cell has been inves-tigated by Watson et al. [62]. Little et al. [63] used thetechnique for sequencing multiply charged proteins andoligonucleotides. The fragments from IRMPD are sim-ilar to those formed by collisional activation.

Martin et al. published a thorough report on peptidesequence analysis using IRMPD [30]. They achievedsub-femtomole peptide sequence analysis with micro-capillary HPLC columns. Accurate masses from a digestof six standard proteins enabled accurate mass analysis(±10 mDa). The authors also successfully demonstratedthe use of IRMPD as a method for MS–MS analysis of

190 200 210 220 230

a

420410400390380370350340 m/z

rel. Int.

383 384 385 386 387m/z

rel. Int.

b

470 520 560 600m/z

rel. Int.

c

680 700 740 760m/z

rel. Int.

720

d

rel. Int.

m/z180

l t

360

rel. Int.rel. Int.

470 520 560 600m/z

470 520 560 600m/z 680 700 740 760

m/z720680 700 740 760

m/z720

rel. Int.

m/z

Fig. 6 Gas-phase products from reaction of a-pinene with ozone;a represents a number of reaction products which have beenidentified, most of them contain at least one carboxylic functionalgroup, b represents dimers of the carboxylic acids, c trimers, andd the range where tetramers appear; the insert shows that at onenominal mass more than one signal appear. Accurate mass dataenable calculation of the elemental composition of the signals withan average error of less than 2 ppm

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an immunopurified mixture of phosphatase regulatoryproteins. By using SDS–PAGE and silver staining ofimmuno-affinity-purified phosphatases they found over100 distinct bands ranging between 15 and 125 kDa.From this mixture a tryptic digest was separated on-lineby HPLC and the recorded MS–MS spectra were sear-ched against the SEQUEST database. The resultsshowed that high-accuracy mass data enabled identifi-cation of differences between sequences of proteins witha mass difference of only some ten millimass units, forexample substitution of glutamine (Q) for lysin (K)(Dm=36 mDa) or phenylalanine for oxidized methio-nine (Dm=33 mDa), or between phosphorylated andsulfated peptides (Dm=10 mDa).

Li et al. [27] also demonstrated the use of IRMPD forpeptide mapping and showed the advantages of LC–FTICR analysis for this task. Results from separation ofa standard mixture of five different proteins are shown inFig. 7. The TIC obtained from separation of the intactproteins is shown at the top; the lower TIC shows the

LC–IRMPD mass spectra. It should be noted that al-though the chromatograms appear the same, the insertsshow the spectra are different, because of the greaterfragmentation of the ions. In comparison, HPLC sepa-ration is faster, simpler, and enables transfer of a largeramount of protein to the MS than is possible using a 2Dgel; this makes LC–FTICR a very useful tool.

Electron-capture dissociation (ECD)

Electron-capture dissociation is a method that uses anelectron beam to fragment ions [64]. Although limited toions that at least contain two charges, ECD results infragmentation different from that obtained by CAD orIRMPD methods. Although the efficiency of productionof fragments by ECD is approximately one third that ofCAD the biggest advantage is that ECD provides moreextensive sequence information from peptides. Frag-mentation using ECD leads to different bond breaksthan in collision activation or IRMPD, thus giving dif-ferent information about a sequence, especially forprotein analysis; this therefore complements the othertechniques very well. Zubarev et al. [65] recently de-scribed the mechanism of ECD in detail.

The use of ECD was realized in parallel by the groupsof Palmblad [66] and Davidson [67], who implementedthis approach for analysis of standard peptides.

Palmblad et al. [66] used LC–FTICR with a novelelectron-injection system employing a micro-capillarycolumn and a sheathless ESI interface to couple LC to a9.4-T FTICR instrument. The authors obtained ECDspectra from a mixture of seven standard proteins afterchromatographic separation. ECD fragmentation en-abled partial sequencing of substance P. They also re-ported results obtained from a BSA tryptic digest, inwhich they could identify 15 of the 75 predicted trypticpeptides on the time-scale of the chromatographic run.

Davidson et al. [67] used the ECD technique for di-rect LC–FTICR analysis of pepsin-digested peptides.They were able to sequence residues 22–104 from a di-gest of cytochrome C—residues 1–22 gave a weak signalwith and without ECD. The authors concluded thatpepsin is probably not the best choice for peptidedigestion in combination with ECD fragmentation.They assumed that trypsin is favored because it leads tosuperior fragmentation.

Multipole storage-assisted dissociation (MSAD)

Another method, in which all ions are fragmented insidean RF-multipole located after the transfer capillary inthe ion source, and is thus independent of analyzercharacteristics, is described as multipole storage-assisteddissociation (MSAD) [68]. This kind of fragmentation,of all ions, is induced without further separation ofprecursor ions by employing extended ion accumulationintervals in this higher-pressure region. The pressure in

Fig. 7 Two LC–FTICR TIC obtained from separation of fiveknown proteins. Top: TIC of intact proteins, the insert shows amass window spanning the isotopic distribution for a protein ofaverage mass 8,564.9 Da. Bottom: TIC of the same samplerecorded in IRMPD mode; the two TICs were detected onalternate scans during the same HPLC run. (Reprinted withpermission from [27]. Copyright [1999] American ChemicalSociety)

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this region enables fragmentation of stable ions beforetransfer to the cyclotron cell.

For the identification of different human liver diace-tyl reductases Tanaka et al. [69] achieved fragmentationby accumulating ions inside the hexapole. They used analternating acquisition approach by switching fromnormal standard acquisition conditions to in-sourcefragmentation conditions from scan to scan. This en-abled normal and a fragmented spectra to be obtainedevery other scan. At the end two different data files werecreated; the odd scan numbers contained the normaldata set and the even scan numbers contained the frag-mented scans.

The mass accuracy and mass resolution from theFTICR minimizes false matches from peptide libraries.Combination with a nano-LC enabled separation andidentification of three diacetyl reductases, which corre-spond to approximately half of the total diacetylreducing activity inside the human liver.

CE–FTICR applications

Even more difficult than combining LC with MS iscoupling with CZE. Although it is not the main topic ofthis review some brief examples of the work that hasbeen done should be mentioned here.

The problems of coupling the two techniques aremainly because of the characteristics of CZE, in whichvery small amounts of sample (lower nanolitre range) offairly high concentrations are injected. This often makesit necessary to have highly concentrated samples avail-able.

Valaskovic et al. [70] investigated, among othersamples, a single red blood cell. They reported thedetection of �7 amol of human carbonic anhydrase.Although an accurate relative molecular mass wasavailable, that data did not match those in a proteindatabase. Collisional activation in the nozzle-skimmerregion gave data enabling explanation of a 42-Da dif-ference resulting from acetylation.

Hofstadler et al. [35, 36] reported the analysis of alysate from a single human erythrocyte. The results re-vealed the presence of both the a and b chains of humanhemoglobin.

Wetterhall et al. [71] published one of the rare papersthat does not use model or standard samples for acoupling to FTICR. The authors used the combinedhigh-resolution capabilities of both FTICR and CZE forinvestigation of human cerebrospinal fluids (CSF), aphysiological fluid that is produced by choroid plexus inthe ventricles of the cerebral hemisphere of the brain.The authors presented results from proteomic analysisof a tryptic digest from CSF proteins. CZE–FTICRanalysis enabled identification of twice as much proteinas was identified by direct infusion experiments. Withthese data 30 proteins were identified with 95% confi-dence and an error of less than 5 ppm [71].

Conclusion

LC–FTICR is a fascinating and powerful analyticaltechnique for analysis of highly complex mixtures, a fieldthat is just beginning to grow.

Although published reports of the use of LC–FTICRon ‘‘real’’ samples or in quantification experiments arevery rare, the possibilities afforded by this combinationhave been shown.

Although FT MS has exceptional mass accuracy andmass resolution capabilities, which can sometimeseliminate the need for further separation by a chro-matographic technique, there are many problems inproteomics, biotechnology, and combinatorial or envi-ronmental chemistry that need additional separationbefore MS analysis.

It has been shown that the performance of FTICR issuperior at lower flow rates, because a large gas loadreduces the performance of this ultra high-vacuum sys-tem. Coupling to nano-LC thus seems a perfect match,increasing both sensitivity and accuracy. One criticalfactor so far has been the loading of the cyclotron cell.Too many ions in the cell lead to space-charge effectsthat reduce the performance of the system. Now, withthe introduction of hybrid Q-q-FTICR systems, it seemsthat this can be handled very elegantly.

Technical improvements of the techniques of com-mercial FT MS have advanced this method to a levelwere it can be used almost routinely [72], althoughsometimes one wishes software improvements wouldmatch hardware improvements to make it more userfriendly.

Is FTICR the method of choice for the coupling withLC? Certainly not always and, as has been demon-strated, sometimes the added dimension of LC separa-tion does not reveal more information than a simpleinfusion experiment.

Hau et al. [73] compared a Q-q-TOF instrument withan FTICR in regard of mass accuracy and resolutionand found that although the resolution (�10,000) andaccuracy of Q-q-TOF are sufficient for routine deter-minations of elemental composition, the biggest advan-tage of the FTICR is the long-term stability of theinstrument. Whereas other instruments have theadvantage of lower price, LC–FTICR still has superiorcapabilities.

Acknowledgements The authors want to thank Dr Jorg Hau (NestleResearch Center) for helpful discussions. We also want to thank DrMathias Witt (Bruker Daltonics, Bremen) and Dr Reinhold Pesch(Thermo, Bremen) for supplying some of the graphics.

References

1. Baldeschwieler JD (1968) Science 159(3812):2632. Gross ML, Wilkins CL (1971) Anal Chem 43(14):A653. Comisarov MB, Marshall AG (1974) Chem Phys Lett

25(2):282–283

1023

4. Comisarov MB, Marshall AG (1974) Chem Phys Lett26(4):489–490

5. Hughey CA, Rodgers RP, Marshall AG (2002) Anal Chem74(16):4145–4149

6. Whitehouse CM, Dreyer RN, Yamashita M, Fenn JB (1985)Anal Chem 57(3):675–679

7. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM(1989) Science 246(4926):64–71

8. Morris HR, Paxton T, Dell A, Langhorne J, Berg M, BordoliRS, Hoyes J, Bateman RH (1996) Rapid Commun MassSpectrom 10:889–896

9. Pinto DM, Boyd RK, Volmer DA (2002) Anal Bioanal Chem373(6):378–389

10. Marshall AG, Hendrickson CL, Jackson GS (1998) MassSpectrom Rev 17(1):1–35

11. Marshall AG, Hendrickson CL (2002) Int J Mass Spectrom215(1–3):59–75

12. Alford JM, Williams PE, Trevor DJ, Smalley RE (1986) Int JMass Spectrom Ion Proc 72:33–51

13. Caravatti P (1990) USA Patent 4,924,08914. Senko MW, Hendrikson CL, Emmett MR, Stone DH Chi,

Marshall AG (1997) J Am Soc Mass Spectrom 8:970–97615. Beu SC, Laude DA (1991) Int J Mass Spectrom Ion Proc

104(2):109–12716. Beu SC, Senko MW, Quinn JP, McLafferty FW (1993) J Am

Soc Mass Spectrom 4(2):190–19217. Douglas DJ, French JB (1992) J Am Soc Mass Spectrom 3:398–

40818. Douglas DJ USA Patent 5,179,27819. Xu HJ, Wada M, Tanaka J, Kawakami I (1993) Nucl Instrum

Methods A 333:274–28120. Belov ME, Anderson GA, Angell NH, Shen YF, Tolic N,

Udseth HR, Smith RD (2001) Anal Chem 73(21):5052–506021. Belov ME, Rakov VS, Nikolaev EN, Goshe MB, Anderson

GA, Smith RD (2003) Rapid Commun Mass Spectrom17(7):627–636

22. Schwarz JC, Zhou X-G, Bier ME (1996) US Patent 14,572,02223. Stockton GW, Meek JT, Millen WG, Wayne RS (1991) In:

Asamato B (ed) FT-ICR/MS: analytical applications of Fou-rier-transform ion cyclotron resonance mass spectrometry.VCH, New York, pp 235–272

24. Blakely CR, Carmody JJ, Vestal ML (1983) J Am Soc Chem102(18):5931–5933

25. Aberth W, Straub KM, Burlingame AL (1982) Anal Chem54(12):2029–2034

26. Emmett MR, White FM, Hendrickson CL, Shi SDH, MarshallAG (1998) J Am Soc Mass Spectrom 9(4):333–340

27. Li WQ, Hendrickson CL, Emmett MR, Marshall AG (1999)Anal Chem 71(19):4397–4402

28. Quenzer TL, Emmett MR, Hendrickson CL, Kelly PH, Mar-shall AG (2001) Anal Chem 73(8):1721–1725

29. Chowdhury SK, Katta V, Chait BT (1990) Rapid CommunMass Spectrom 4(3):81–87

30. Martin SE, Shabanowitz J, Hunt DF, Marto JA (2000) AnalChem 72(18):4266–4274

31. Peng J, Gysi SP (2001) J Mass Spectrom 36:1083–109132. Yates JR (1998) J Mass Spectrom 33:1–1933. Shen YF, Zhao R, Belov ME, Conrads TP, Anderson GA,

Tang KQ, Pasa-Tolic L, Veenstra TD, Lipton MS, Udseth HR,Smith RD (2001) Anal Chem 73(8):1766–1775

34. Shen YF, Tolic N, Zhao R, Pasa-Tolic L, Li LJ, Berger SJ,Harkewicz R, Anderson GA, Belov ME, Smith RD (2001)Anal Chem 73(13):3011–3021

35. Hofstadler SA, Severs JC, Smith RD, Swanek FD, Ewing AG(1996) Rapid Commun Mass Spectrom 10(8):919–922

36. Hofstadler SA, Swanek FD, Gale DC, Ewing AG, Smith RD(1995) Anal Chem 67(8):1477–1480

37. Tang KQ, Tolmachev AV, Nikolaev E, Zhang R, Belov ME,Udseth HR, Smith RD (2002) Anal Chem 74(20):5431–5437

38. Fuchser J, Witt M, Baykut G (2001) In: Proceedings of the 49thASMS conference mass spectrometry and allied topics, Chicago

39. Horning S, Malek R, Wieghaus A, Senko MW, Syka JEP(2003) In: Proceedings of the 51st ASMS conference massspectrometry and allied topics, Montreal

40. Null AP, George LT, Muddiman DC (2002) J Am Soc MassSpectrom 13(4):338–344

41. Kjeldsen F, Haselmann KF, Sorensen ES, Zubarev RA (2003)Anal Chem 75(6):1267–1274

42. Stockton GW, Millen WG, Meek JT (1991) In: Asamato B (ed)FT-ICR/MS: analytical applications of Fourier-transform ioncyclotron resonance mass spectrometry. VCH, New York, pp273–291

43. Stacey CC, Kruppa GH, Watson CH, Wronka J, Laukien FH,Banks JF, Whitehouse CM (1994) Rapid Commun MassSpectrom 8(7):513–516

44. Marotta E, Lapolla A, Fedele D, Senesi A, Reitano R, Witt M,Seraglia R, Traldi P (2003) J Mass Spectrom 38(2):196–205

45. Ihling C, Berger K, Hofliger MM, Fuhrer D, Beck-SickingerAG, Sinz A (2003) Rapid Commun Mass Spectrom17(12):1240–1246

46. Witt M, Fuchser J, Baykut G (2003) J Am Soc Mass Spectrom14(6):553–561

47. Fruchtel JS, Jung G (1996) Angew Chem Int Ed 35(1):17–4248. Jung G (ed) (1996) Combinatorial peptide and non-peptide

libraries. Verlag Chemie, Weinheim49. Reetz MT (2001) Angew Chemie Int Ed 40(2):284–31050. Schmid DG, Grosche P, Jung G (2001) Rapid Commun Mass

Spectrom 15:341–34751. Went FW (1960) Nature 187:641–64352. Andreae MO, Crutzen PJ (1997) Science 276:1052–105853. Ravishankara AR (1997) Science 276:1058–106554. Schrader W, Geiger J, Godejohann M, Warscheid B, Hoffmann

T (2001) Angew Chem 113:4129–4132; Angew Chem Int Ed40:3998–4001

55. Geiger J, Korte EH, Schrader W (2001) J Chromatogr A922:99–110

56. Warscheid B, Hoffmann T (2002) Rapid Commun MassSpectrom 16(6):496–504

57. Yu J, Crocker DR III, Griffin RJ, Flagan RC, Seinfeld JH(1999) J Atmosph Chem 34:207–258

58. Schrader W, Geiger J, Hoffmann T, Klockow D, Korte EH(1999) J Chromatogr A 864:299

59. Kuckelmann U, Warscheid B, Hoffmann T (2000) Anal Chem72:1905–1912

60. Gauthier JW, Trautman TR, Jacobson DB (1991) Anal ChimActa 246(1):211–225

61. Sannes-Lowery K, Griffey RH, Kruppa GH, Speir JP, Hof-stadler SA (1998) Rapid Commun Mass Spectrom 12(23):1957–1961

62. Basov NG, Markin EP, Oraevski AN, Pankratov AV, Shach-kov AN (1971) JETP Lett 14:165–169

63. Watson CH, Baykut G, Eyler JR (1987) 59(8):1133–113864. Little DP, Speir JP, Senko MW, O’Connor PB, McLafferty FW

(1994) Anal Chem 66(18):2809–281565. Kruger NA, Zubarev RA, Horn DM, McLafferty FW (1999)

Int J Mass Spectrom 187:787–79366. Zubarev RA, Haselmann KF, Budnik B, Kjeldsen F, Jensen F

(2002) Eur J Mass Spectrom 8(5):337–34967. PalmbladM, TsybinYO,RamstromM, Bergquist J, Hakansson

P (2002) Rapid Commun Mass Spectrom 16(10):988–99268. Davidson W, Frego L (2002) Rapid Commun Mass Spectrom

16(10):993–99869. Tanaka Y, Sato I, Iwai C, Kosaka T, Ikeda T, Nakamura T

(2001) Anal Biochem 293(2):157–16870. Valaskovic GA, Kelleher NL, McLafferty FW (1996) Science

273(5279):1199–120271. Wetterhall M, Palmblad M, Hakansson P, Markides KE,

Bergquist J (2002) J Proteome Res 1(4):361–36672. Wesener JR, Lagojda A (2001) In: Proceedings of the 49th

ASMS conference mass spectrometry and allied topics, Chicago73. Hau J, Stadler R, Jenny TA, Fay LB (2001) Rapid Commun

Mass Spectrom 15(19):1840–1848

1024