The multiply charged ions produced by electrospray ionization of peptides of molecular masses up to 29 kDa have been successfully introduced into a Fourier transform mass spectrometer of unique capabilities for tandem mass spectrometry, large ion dissociation, and resolution. Electrospray ionization places an unusually high number of charges on a peptide yielding mass/charge (m/z) values of 600-1500; in this range at normal operating pressures (-10-9 torr; 1 torr = 133.3 Pa) Fourier-transform mass spectrometry resolving power is >100,000. Although only 10-torr pressure has been obtained with the initial interface, the resulting resolving power of 5000 makes possible the resolution of isotopic peaks of multiply charged ions. Mass measuring accuracies of a few daltons for molecular masses up to 17 kDa have also been achieved.Of the ionization methods that have revolutionized the applicability of mass spectrometry to large molecules, electrospray (1-10) has the unusual advantage of producing ions with by far the highest relative number of charges. This was first demonstrated by Fenn and coworkers (4-6), based on the pioneering work of Dole and coworkers (3), with very recent exciting applications to proteins and nucleotides with molecular masses up to 130 kDa by Smith, Loo, and coworkers (7,8), and with highly accurate mass measurements by the Fenn (6), Smith (8), and Henion (9, 10) groups. For even the largest proteins, molecular ions with mass/charge (m/z) values of 600-1400 can be obtained with minimal fragmentation, making it possible to use a quadrupole mass spectrometer with correspondingly small upper mass limits (4-10). In considering alternative instrumentation, Fouriertransform mass spectrometry (FTMS) exhibits resolution that is uniquely high, although inversely proportional to the m/z value, with resolving power values of >100,000 reported for this m/z range (11-13). Increasingly large molecules require an exponential increase in the amount of information necessary for structural characterization; here, the extra data dimensions of tandem mass spectrometry (MS/MS, MS') are extremely promising (14,15) analyzer over such a large mass range for each selected primary ion. On the other hand, the pressure requirement for high-resolution FTMS is much more stringent than for most other mass spectrometers (=10-9 torr vs. -.-10-6 torr; 1 torr = 133.3 Pa), an obvious disadvantage for the atmosphericpressure electrospray process. This paper reports successful FTMS spectra by using electrospray ionization for peptides and proteins of molecular masses up to 29 kDa, although ion source pressures of only 10-7 torr have been achieved with this preliminary system. EXPERIMENTALThe Virginia FTMS instrument (19,22,23) has a 7 T magnet and a 2.8 x 2.8 x 7.8 cm ion cell, with ion introduction through 14, 25, and 86 cm rf-only (1.25, 0.87, 0.87 MHz) quadrupoles, each with an HV-202-6C 680 liters/s cryogenic pump. Electrospray (and "electrosplotch") ionization used gravity feed through a 900 bent syringe tip (500-,ul H...

Peptide molecular ion species up to m/z 3055 introduced into a Fourier-transform mass spectrometer can be made to undergo extensive fragmentation by electrically floating the ion cell. The proportion of ions dissociated increases with increasing voltage, with 48 eV producing the highest absolute abundance of fragment ions above m/z 200. At this energy, spectra closely resemble those from photodissociation at 193 nm, indicating an internal energy deposition of 6-7 eV; change of product abundances with kinetic energy resembles a conventional breakdown curve. The precursor ions apparently are electrostatically attracted to strike screen wires across the ion cell entrance, producing daughter ions of low kinetic energy.

Electrospray Ionization with Fourier-Transform Mass Spectrometry. Charge State Assignment from Resolved Isotopic Peaks.Matrix-assisted laser desorptionl and electrospray applicability of mass spectrometry to compounds of molecular weight >100,000, a revolutionary advance. Of electrospray advantages, its ions, even of mass 130,000,4 show m/z values of 600-1600, within the m/z range of instruments such as the q~a d r u p o l e~-~ and Fourier-transford mass spectrometers; also, molecular weight measurements on relatively large molecules are surprisingly accurate (-1/104) . 3-7 challenges are to obtain molecular weight information from more complex mixtures and structural information from the dissociation of individual molecular ion species .7-13 Such tandem mass spectrometry (MS/MS)8 has been shown, for example, to provide relatively complete amino acid sequence information for mixtures of oligopeptidesg of masses up to 2500,1° but the exciting recent MS/MS results from electrospray ionization5s7 delineate two challenging problems. First, to determine the mass (m) a value must be assigned to z , which can be done easily for relatively pure compounds giving a very few values of m i , each of which gives a multitude of mi/z values. individual isotopic peaks, the number of these in an m/z unit unequivocally defines the number of charges, without the necessity to identify other mi/z peaks. Secondly, the further dissociation of fragment ions can give further information concerning their isomeric using further mass analyses (MS"). '' The Fourier-transform (FT) instrument with its unusually high resolving power and MS" capabilities is a promising solution to both problems.6 FTMS, in collaboration with Hunt and Shabanowitz ,6 obtained spectra for compounds of molecular weights up to 29,000, but with relatively poor resolution, presumably because ion cell pressures were torr. This preliminary report describes results from an FTMS instrument with a more efficiently pumped interface yielding spectra from much lower quantities of larger molecules, but exhibiting an unexpected inverse effect of mass on the resolving power obtained For peaks of similar m/z values. extend the Exciting furtherWe have pointed out6 that with resolution sufficient to resolveThe original successful combination of electrospray with Experimental. Electrospray generated ions from H20/MeOH/HOAc solution are conducted by quadrupole rods through four stages of differential pumping to the dual ion cell of a prototype Nicolet FTMS-200014,15 (Figure 1). Ions are admitted to the grounded cell for -7 ms, the trapping plates raised to +8 V , the ions allowed to relax for 45 s , 1 4 and spectra measured with 500 KHz bandwidth and 32 K data points.Results and discussion. Normal high resolution FTMS measurements, such as 833,000 at m/z 35716 and 160,000 at m/z 3200,'' require ion cell pressures below 5 x lo-' torr was achieved in full electrospray operation with the Figure 1 system. However, ion transfer through the 2 mm conductance limit was poor, s o this was rem...

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Unit-resolution mass spectra have been obtained for peptides as large as 17 kDa, providing information on impurities and adduct ions, as well as accurate molecular weight values. Electrospray ionization produces many multiply-charged species of the same mass; isotopic peak resolution provides direct charge state assignment from the unit mass spacing of the isotopes. This is of special value when the spectrum also has many masses, such as from precursor ion dissociation or impurities. Mass measuring errors not only are concomitantly lower (<0.1 Da) than when the isotopic peaks are unresolved but also are independent of variations in '3C/12C natural isotopic abundances. Also, larger errors are avoided that occur when the measured peak envelope includes impurity or adduct ions. This also benefits tandem mass spectrometry; dissociation of peptide ions as large as 8.5 kDa yields fragment masses consistent (<0. (21)(22)(23)(24)(25)(26) can be used to measure simultaneously all such ions as large as 17 kDa with 50,000-80,000 resolving power and, by using an internal standard, with <0.1 Da (<6 ppm) mass errors. As reported here, this enhanced high-resolution capability is also valuable for characterizing impurities (6-8) adducts (9-11) and, by tandem mass spectrometry (MS/MS) (3,7,8,(12)(13)(14)(15), amino acid sequences.EXPERIMENTAL PROCEDURES Radio frequency-only quadrupole lenses and five stages of differential pumping are used to transport the ions electrosprayed at -103 torr to the FTMS ion-measurement cell at <10-8 torr in a 2.8-T magnet. A pulse of N2 gas is admitted to the ion cell to cool the ions translationally before measurement; other experimental details were as given (21-23).RESULTS AND DISCUSSION and the measured value (weighted average of isotopic peak values; external standard: gramicidin S, Mr = 1141) is 12,352.20 (12,358.22 for the most abundant isotopic peak).Note that a 5-per-mil shiftt in the 13C/'2C ratio would change the isotopically averaged molecular weight (centroid of the unresolved isotopic peaks) (1-18) by 0.3 Da but would only affect the abundances of the isotopic peaks, not their masses. These experimental procedures have not produced, to date, high-resolution ESI spectra of molecules as large as albumin (66 kDa), although its spectrum (Fig. 2) was improved (22).By using a quadrupole mass spectrometer (6, 7), the ESI mass spectra of bovine ubiquitin, mMr 8559.62, showed peaks of measured masses 114.7 Da (6) and 115.1 Da (7) below those of the (M + nH)n+ ubiquitin peaks; § this was postulated (7) to result from an impurity missing the two carboxyl-terminal glycines, which would lower the mass by 114.04 Da. The ESI/FTMS mass spectrum of the same sample (7) (Fig. 3) shows an mMr of 8559.45 and peaks presumably from the same impurity at a mMr of 8445.43 (Am = 114.02). Additional anomalous peaks not resolved in the quadrupole spectra (6,7) give an average mMr of 8623.39 (Am = 63.94); replacing a valine by a tyrosine would increase the molecular weight by 63.99. As noted (23), ident...