Structural characterization of protein tryptic peptides via liquid

Beryl M. Tracey and David E. G. Shuker .... Gloria M. Sheynkman , Michael R. Shortreed , Anthony J. Cesnik , Lloyd M. Smith ..... Anita Saraf , John R...
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Anal. Chem. 1991, 63,1193-1200

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Structural Characterization of Protein Tryptic Peptides via Liquid ChromatographyIMass Spectrometry and Collision-Induced Dissociation of Their Doubly Charged Molecular Ions Thomas R. Covey* Sciex, 55 Glen Cameron Road, Thornhill, Ontario, Canada L3T lP2 Eric C. Huang’ and Jack D. Henion* Drug Testing and Toxicology Program, Cornell University] 925 Warren Drive, Ithaca, New York 14850

The formatlon of multlply charged molecular Ions vla the tkld-asdsted Ion evaporatkn machanism durlng electrogpray lonlzatlon enables the u w of an atmospheric pressure lonlzatlon qUadNpd@mass spectrometer system for characterking Mokgkally Important peptldes. The stralghtforwardknplementatlon of hlgh-performance llquld chromatography (HPLC) Into thls new strategy to determlne the molecular welght of tryptlc peptides vla the pneumatlcally assisted electtospray (Ion spray) Interface Is presented. Examples u#zlng both “ b o r e (1.0 mm) and standard bore (4.6 mm) Inside diameter colmm are shown for the LC/MS molecular weight determlnatlon of tryptlc peptldes In methlonyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptk d t g d onto 1mm 1.d. HPLC columns provkled full-scan LC/MS or LC/MS/MS results wlthout postcolumn spllttlng of the effluent. When standard 4.6 mm 1.d. HPLC columns were used, a 20:l postcolumn spllt was utlllzed, whlch requlred from 1 to 5 nmol of Injected tryptlc dlgest for full-scan LC/MS or LC/MS/MS results. Colllslon-Induced dlsroclatlon (CID) mass spectra resultlng from elther “lnfudon” or on-llne LC/MS/MS analysls of the abundant doubly charged Ions that predomlnate for tryptic peptldes under ektroapray condltlons provlded structurally useful 88quence lnformatlon for met-hGH and human hemoglobln trvptlc Ugests. The dower maaa rpectrometer scan rate used durlng lnfwlon of sample provkles more accurate mass asdgnments than on-llne LC/MS or LC/MS/MS, but the latter on-Wne expwlmentr pmcluck amblguttks caused by matrlx or component Interferences. However, In m e Instances very weak CID product Ions preclude complete tryptic peptlde structural characterlzatlon based upon the CID data alone. The on-llne LC/MS/MS analysls of the tryptic dlgest from human hemoglobln normal &chaln provlded sufflclent overlapping structural lnformatlon to deduce the sequence of a representative tryptlc fragment. Thls approach provldes an effective means of characterlzlng these blokgkally Important compounds.

INTRODUCTION Application of mass spectrometry to the characterization of compounds with molecular weights greater than lo00 was rare prior to the development of desorption ionization methods. These developments included field desorption ( l ) , *Towhom correspondence may be addressed. Current address: Merck Sharp & Dohme Research Laboratories,

West Point, PA 19486.

0003-2700/91/0383-1193$02.50/0

fast atom bombardment ( 2 , 3 )and plasma desorption (4-7). More recently, laser desorption ( 8 , 9 )and electrospray ionization (10-12) processes have appeared as new technology for high-mass analysis by mass spectrometry. The “state of the art” of mass spectrometry for the characterizationof biological molecules was recently reviewed (13). Fenn and co-workers were the first to describe the analysis of samples containing polymers, peptides, and proteins with molecular weights beyond 20 000 using an electrospray interface to affect electrospray ionization a t atmospheric pressure on a quadrupole mass spectrometer (14, 15). Two other groups immediately duplicated these initial observations (11,16). These early results clearly indicated that the existing limitations in mass range for high molecular weight analytes can be elegantly surmounted by utilizing an ionization technique that increases the number of charges on the molecule thereby bringing the m / z value into the available working range of quadrupole mass analyzers. Electrospray ionization a t atmospheric pressure has recently been demonstrated on Fourier transform (17) and magnetic (18) mass analyzers, which opens exciting possibilities for further mass range extension and greater mass accuracies. One model for electrospray ionization is described as field-assisted desorption of solute ions from rapidly evaporating liquid droplets (19). Another scenario proposes an electrohydrodynamic disintegration process (20). Whichever model bears most resemblance to the precise physical phenomena, the technique, in comparison to other soft ionization methods, appears capable of generating extensive multiple charging of large molecules. The use of simple equations on the sequence of multiply charged ions, formed by proton, ammonium, or allrali-metal ion adducts enables the calculation of the number of charges and the deduction of the parent maw ( 1 1 , 21). Like the electrospray interface (22),the ion spray interface (23)produces gas-phase ions via the ion evaporation mechanism (19) from a flowing liquid stream, whereupon nebulization and ionization occur at atmospheric pressure. Both techniques produce the same mass spectra and are ideal candidates for coupling chromatographic separation systems with atmospheric pressure ionization mass spectrometry. The ion spray system utilizes pneumatic nebulization in addition to an electric field to improve the stability and utility of the device for LC/MS operation. Pneumatic dispersion of the droplets stabilizes the ion current over a broad range of liquid flow rates and allows the use of mobile phases with high surface tension, such as those having a high aqueous content. Several LC/MS applications have demonstrated the utility of this method as a generally applicable LC/MS interface (24). Reversed-phase HPLC is the method of choice for the analysis of mixtures containing small proteins and peptides 0 1991 American Chemical Soclety

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Curlain On because it routinely delivers good separation efficiency and offers the possibility of isolating purified individual peptides lon for subsequent characterization by amino acid analysis, sespy 5kv quencing, or mass spectrometric analysis. The capability for imm.2.imm,or4.6 mm HPLC Column on-line LC/MS molecular weight determination of peptides from the enzymatic digestion of proteins adds efficiency to the procedure by minimizing sample handling losses and reducing the time required for analysis. Approaches using continuous-flow FAB (25-27) and thermospray (28,29) have also been implemented for the analysis of peptide mixtures. Recent reports suggest the ion spray LC/MS interface is another promising tool for on-line LC/MS determination of peptides and proteins (30-32). Both low-energy (33,34) and high-energy (13d,e, 35,36) Figure 1. Schematic diagram of the ion spray interfaceIAP1source collision data have demonstrated that collision-induced diswith postcolumn splitter. The split llne k composed of 100 pm 1.d. sociation (CID) mass spectra provide important sequence fused silica adjusted in length to obtain the required split ratio. information. The use of tandem mass spectrometry (MS/MS) to determine the amino acid sequence of the peptides prowithout an intermediate differentially pumped region. The atduced from enzymatic digestion of proteins has been utilized mospheric side of the sampling cone was bathed with a curtain in conjunction with classical biochemical techniques (34,s).of high-purity dry nitrogen gas (0.7 L/min), which acted both as a barrier to restrict contaminantsand solvent vapor from entering Recent data have demonstrated potential advantage3 available the mass spectrometer vacuum and as an ion/molecule declusfrom the use of the multiply charged parent ions characteristic tering region. High vacuum was maintained by cryogenically of electrospray ionization for fragmentation in low-energy cooled surfaces maintained at 15-20 K. triple-quadrupole systems (37-40). For LC/MS experiments the mass spectrometer was operated In this report the implementation of LC/MS for molecular under unit-mass resolution conditions. The mass calibration weight determination of peptides from tryptic digests of procedure is based on the ammonium adducts observed for the methionyl-human growth hormone (mt-hGH) and human oligomers in a mixture of polypropylene glycol lo00 and 2000 (1 hemoglobin normal &chain are presented by using a tripleX lV M in 80/20 acetonitrile/H20 1mM NH40Ac). During the quadrupole mass spectrometer equipped with an atmospheric on-line chromatographicanalyses mass spectra were scanned from pressure ionization (API)ion source and an ion spray LC/MS 300 to 2400 Da at 4 s/scan. For the MS/MS experiments involving infusion sample introduction quadrupoles 1and 3 were operated interface. Various operational characteristics of the system at constant resolution with a mass peak width at half-height equal are described including adaptation of the system to convento 2 u. The precursor ion was focused by Q-1 while 6 - 3 was tional 4.6 mm i.d. as well as 1 mm i.d. microbore HPLC scanned from m / z 300 to 2400 over a 25-s time period. Under columns. The benefits and limitations of CID mass spectra these mass resolution conditions the product ion mass spectra obtained from the infusion of typical protein tryptic digests contain polyisotopic clusters of peaks. The bar graph plots for are also contrasted with those obtained from on-line HPLC CID mass spectra are plotted in these instances with the bar experiments. spacings equal to 0.1 u because the acquisition algorithm sampled ion current at 0.1-u step&Thus the CID bar graphs show multiple EXPERIMENTAL SECTION 'peaks" in these experiments. In the full-scanLC/MS acquisition mode the acquisition algorithm sampled ion current in 0.511 Liquid Chromatography. The liquid chromatugraphic system steps, consisted of an AB1 Model 140A dual-syringe pump (Foster City, and the bar graph line widths are 0.5-u spacings in these instances. CA) for the microbore work and a Perkin-Elmer 250 gradient This sampling rate enables detection of doubly charged ions system (Norwalk, CT) for the conventional-bore HPLC separaoccurring at nonintegral masses. The electron multiplier detector tions. A Rheodyne Model 7410 injection valve equipped with a was operated in this instrument's normal pulse-counting mode. 10-pLloop (Cotati, CA) was used with both systems. Microbore The daughter ion mass spectrawere obtained either by infusion separations were accomplished with a 1-mm X 100-mm Aquapore of a solution of the protein tryptic digests at a flow of 2 pL/min C18 column (ABI) at a flow of 40 pL/min with no postcolumn or by on-line LC/MS/MS analyses of the tryptic digests. The splitting of the effluent, while conventional HPLC separations on-line results were achieved either by using 1 mm i.d. packed were performed with a 4.6-mm X 100 mm column with the same columns with no postcolumn split, or 4.6 mm i.d. columns with stationary phase and eluent maintained at 1mL/min and a 201 a postcolumn split of 201. All CID mass spectra were obtained postcolumn split. A linear gradient was used for the LC/MS on the doubly charged precursor ions at a collision energy of 100 separations beginning with 100% HzO followed by increasing eV (50 V acceleratingpotential on the collision cell). Argon was acetonitrile content at 1%/min; the concentration of trifluoroused as the collision gas in an open collision cell. The collision acetic acid was constant at 0.1% (v/v). The exit of the chrogas thickness was calculated to be 4.5 X 10" atoms/cm2 corrematographic column was connected directly to the ion spray sponding to an analyzer pressure of 2.1 X lod Torr. Under these LC/MS interface (23) with a 10-cm length of 100 pm i.d. X 175 conditions single collisions occur (19). pm 0.d. fused-silica capillary (Polymicro Technologies, Phoenix, A TAGA 6000E triple-quadrupole mass spectrometer was used AZ). For the postcolumn flow-splitting experiments a Valco for the on-line LC/MS/MS analysis of the tryptic digest of human (Houston,TX)zero dead volume 'tee" was inserted between the hemoglobin normal B-chain. The strategy for characterization column and interface, allowing the split ratio to be adjusted by of tryptic digests by LC/MS/MS was to perform a preliminary varying the length of the fused-silica split line (100 pm i.d.). The LC/MS analysis, which provided the characteristic doubly charged ion spray interface was operated at 5 kV with 40 psi nebulizing parent ion, followed by a subsequent LC/MS/MS analysis, which gas (compressed air) at a flow of 1.0 L/min. A schematic repsubjected each known parent ion of interest to CID. The full-scan resentation of the system is shown in Figure 1. range in these on-line experiments was from m / z 100to 1200. All Mass Spectrometry. A SCIEX API I11 and a TAGA 6OOOE other experimental parameters were as previously reported (33). triple-quadrupole mass spectrometer equipped with a standard Chemicals and Samples. Sequence grade trifluoroacetic acid atmospheric pressure ionization (API) source (Sciex, Inc., (TFA) was obtained from Sigma Chemical Co. (St. Louis, MO), Thornhill, Ontario, Canada) were used to sample ions produced and HPLC grade acetonitrile was obtained from Burdick and from the ion spray interface. In this system (Figure 1)ions were Jackson Laboratories (Muskegon, MI). HPLC grade water was sampled into the vacuum for mass analysis through a 125pm i.d. generated in-house with a Millipore Milli-Q water purification orifice in the end of a core extending toward the atmosphere. The system (Bedford, MA). TPCK-treated Trypsin (T-8642) and atmosphere-to-vacuum transition occurs through this orifice ammonium bicarbonate (A-6141) were obtained from Sigma I

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Table I. Peptides Produced by Digestion of Methionyl Human Growth Hormone with Trypsinn identifier residues [M + HI+ T1 T2 T3 T4 T5 T6b

1-9 10-17 18-20 21-39 40-42 43-65

1061.58 979.50 383.21 2342.14 404.22 2616.24

T7 T8 T10

66-71 7278 79-95 96-116

762.36 844.49 2055.20 2262.13

T11 T12 T13 T14 T15 T16b T17 T18, T19 T17-Tl9 T20C T21C

117-128 129-135 136-141 142-146 147-159 160-168 169 170-179 169-179 180-184 185-192

1361.67 773.38 693.39 626.32 1489.69 1148.55 147.11 1253.62 1381.71 618.34 785.31

TlOcl T10c2

96-100 100-116

T9

1191

'7

sequence MFPTIPLSR LFDNAMLR AHR LHQLAFDTYQEFEEAY IPK EQK YSFLQNPQTSLCFSESIPTPSNR EETQQK

SNLELLR ISLLLIQSWLEPVQFLR SVFANSLWGASDSNVYDLLK DLEEGIQTLMGR LEDGSPR TGQIFK QTYSK FDTNSHNDDALLK NYGLLYCFR K DMDKVETFLR KDMDKVETFLR IVQCR SVEGSCGF

Microbore LC/MS TIC trace (300-2400 u) from a 7Ogmd injection of methGH tryptic digest. The entire effluent (40 pL/min) entered the Ion spray LC/MS interface. Unassigned peaks are due to nontryptic cleavages. For chromatographicconditions, see text. Figure 2.

A 2+

w\'o (M+2H)

TI1 1361

MW

Nontryptic Cleavages 537.27 1743.90

SVFAN LWGASDSNVYDLLK

O[M + H]+ is the calculated molecular weight for the most abundant monoisotopic species. The information in this table was taken from ref 26. The (M + 1)+ is the calculated molecular weight for the most abundant monoisotopic species. bT6 and T16 are disulfide-linked, with a total molecular weight of 3761.77. cT20 and T21 are disulfide-linked,with a total molecular weight of

1-454.5

I

(M+H)+

.

1362.5

500

1500

2000

mh

B

1400.65.

T I2 MW = 772

Chemical Co. (St. Louis, MO) and used without additional treatment. Methionyl-human growth hormone (met-hGH, Protropin) was obtained from Genentech (South San Francisco, CAI. The protein tryptic digestion procedure was performed as described in the literature (26).

RESULTS AND DISCUSSION LC/MS Analyses of Protein Tryptic Digests. Trypsin is an enzyme that specifically hydrolyzes proteins at the carboxy terminal amide bonds of arginine and lysine amino acid residues. These basic residues in addition to histidine and the amino terminus me the moat likely protonation sites under these electrospray conditions. Most tryptic peptides desorb predominantly as doubly charged ions (37), so there is little ambiguity with the determination of tryptic peptide molecular weights using thie approach. However, depending on the structure of the peptide, variations in the mass spectra do occur. For example, the presence of a histidine will add an additional charge, and disulfide bridges linking two tryptic peptides increment the number of charges according to the number of additional basic residues. Also, incomplete digestion may occur, for example, when a proline residue is adjacent to a lysine or arginine residue. Mixed mass spectra of several components in one chromatographic peak are situations that require special Consideration. Since reversedphaee HPLC is a well-awptad technique for analyzing tryptic digests (41), it is desirable to implement MS detection as described here. There have been reports for the electroepray characterization of large biomoldes (42,431,and speculation on the mechanism of electrospray ionization (44),but there has been little information demonstrating practical on-line electroepray LC/MS and LC/MS/MS resulta from enzymatic digests (33,38,45).The following discussion focuses on the interpretation of electrospray ionization mass spectra from

500

low

1500

2000

mh

Figure 3. Electrospray mass spectra for the proteln tryptic peptldes 111 andT12fromtheTICtraceshowninFigve2. Spectrarepresent the average of approximately five scans across the top of the LC

peaks.

the LC/MS analyses of protein tryptic digests with particular reference to met-hGH and human hemoglobin. These observations are supported by the mass spectra obtained for tryptic peptides from many LC/MS analyses in our laboratories including tryptic digests from a-and 8-chains of human hemoglobin, bovine serum albumin, &lactoglobulin A and B, bovine cytochrome c, hen egg white lysozyme, recombinant soluble CD4 receptor (a glycoprotein), and several proprietary proteins ranging in molecular weight from 10OOO to 30 OOO. Figure 2 show the total ion current profile from the gradient (see experimental)microbore LC/MS analysis from the injection of 70 pmol (from a total digest of 50 nmol) of met-hGH tryptic digest with the total effluent (40 pL/min) directed to the ion spray LC/MS interface. In this example all of the expected tryptic peptides (Table I) greater than 300 u are accounted for in the total ion chromatogram (TIC) with a few additional peaks resulting from nontryptic cleavages. Rspresentativepeptide maas spectra from an LC/MS analysis of a protein tryptic digest are shown in Figure 3. The T11

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fragment observed at 32.3 min in Figure 2 produces the mass spectrum shown in Figure 3A, while the T12 fragment observed at 14.1 min in Figure 2 is shown in Figure 3B. If two or more multiply charged ions are present in the mass spectrum (Figure 3A), it is a simple matter to calculate the charge state and thus the molecular weight of an unknown compound (11,21). If only a singly charged ion is present in the mass spectrum, the situation becomes more ambiguous although the ion peak shape or its accurate mass determination can facilitate determining the charge state. However, for tryptic peptides it is reasonable to assume that the base peak will be doubly charged on the basis of the characteristic hydrolysis products of trypsin (11). Inspection of the characteristic ion profile data should show an isotope spacing of 0.5 Da for a doubly charged ion (not shown here). TFA adducts or the common adducts of sodium or ammonium will also reveal the charge state because they will appear as peaks spaced by an amount equal to the difference of the mass of the adduct and the proton it replaced divided by the number of charges on the ion. For example, a doubly charged adduct ion of structure (M+ H + Na)2+will be shifted from the doubly protonated molecular ion (M + 2H)2+by 2212 = 11.0 u (not 2312 = 11.5 u). We do not observe alkali-metal ion adducts in these LC/MS mass spectra due to the "desalting" aspect of on-line HPLC and the high declustering (24j) potential used (50 V). Singly charged ions may be unequivocally distinguished from doubly charged ions by their corresponding CID mass spectra. In contrast to the CID daughter ion mass spectra from singly charged ions the daughter ion mass spectrum of doubly charged parent ions may show "fragment" ions up to twice the mass-to-charge ratio of the parent ion. This approach can be extended to higher charge state molecular ions. For example, a triply charged parent ion can generate singly charged daughter ions up to three times the mass-to-charge of its parent ion. However, there is no guarantee that sufficient high-mass daughter ions will be present in the available mass range to distinguish two multiply charged parent ions. The use of accurate mass measurement and high-resolution features afforded by high-performance sector mass spectrometers may also be utilized to identify charge states of multiply charged ions (18), but these techniques cannot be used on quadrupole mass analyzers. Tryptic peptides of masses leas than approximately lo00 u usually show abundant singly charged as well as doubly charged ions (Figure 3B). As the mass difference between charge sites decreases, the singly charged molecular ion increases in relative abundance. With two adjacent multiply charged ions present, there is little ambiguity in assigning the molecular weight using the integral charge-state criteria (11). A typical mass spectrum for a tryptic peptide containing a histidine residue is shown in Figure 4A, where the doubly and triply charged molecular ions are observed of the T15 and T9 tryptic peptides from the TIC shown in Figure 2. The dominance of the doubly charged ion in histidine-containing peptides may be due to the greater solution basicity of the two terminal charge sites (pK, 1G11)compared to that of histidine (pK, = 7). Even in tryptic peptides where more than one histidine is present the doubly charged ion tends to predominate. However, one may not rely upon the observation of a (M + 3HI3+for a tryptic peptide as an absolute indication of a histidine residue. Exceptions occur where a third charge will be observed on a tryptic peptide even though no histidine is present (Figures 3A, 4B). This is expected with peptides of increasing size and is consistent with the observations cited above that protons can be attached to sites on molecules that are not highly basic. Another case where charge states higher than the doubly charged ion occur is when a disulfide bridge links two tryptic peptides (see discussion below).

-

A

, 750

1250

1000

15W

1750

2000

2250

mi2

B

I

1028.0

2+ (M+2H)

T9 MW=2053

(M+H)+ 2050.0 500

750

1000

1250 mh

1500

1750

2000

2250

Figure 4. Electrospray mass spectra of tryptlc peptides Ti5 and T9 from the TIC shown trace in Figure 2. These mass spectra are the average of approximately five scans across the top of the LC peeks.

IIII mn

Figure 5. Electrospray mass spectrum from a mixture of tryptlc peptkles, T10 and T6-Tl6 (dlsulflde bridged), from the 33.6-mln peak in the TIC trace shown In Figure 2. The ions for the two different components in the mixture are labeled in M, and Ma, respectively. These mass spectra are the average of approximately five scans across the top of the LC peaks.

Mixed-mass spectra having several multiply charged components can appear very complex and determination of the molecular weights of the individual components may appear complicated. The situation is simplified by two important phenomena. First, fragmentation of peptides and proteins in the ion source is for all practical purposes nonexistent with electrospray ionization. All ions observed in a mass spectrum, therefore, can be attributed to molecular ions or adducts and their different charge states. Second, the mathematical relationship that exists for ions of the same component generate recognizable patterns and allow for distinguishing the various constituents using any of several available computer algorithms that facilitate making these assignments. A simple example is the unresolved two-component mixture of tryptic peptides T10 and T6-Tl6 (Figure 5), eluting at approximately 33.5 min in Figure 2. Here the two tryptic peptides, T10 and T6-Tl6, are denoted as MI and MP,respectively, in Figure 5. If one assumes that the base peak in the mass spectrum is a molecular ion of unknown charge state,

ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991

then another ion must exist in the spectrum that will correlate with it and provide a value for an integral charge state. An iterative search of the mass spectrum will yield a mass and charge state list that readily provides an internal check on the molecular weight determination. The remaining unassigned ions are then subjected to the same process to identify the second mass spectrum. The result is identification of the second component molecular weight of 3761 u. With this technique mass spectra with greater than 10 multiply charged components have been deconvoluted manually, although the process is easily automated by computer algorithms. Ironically, as the complexity of the mass spectrum increases due to the presence of higher charge states for each component, the ability to identify individual components also increases. Confidence in identifying the masses of individual components in complex mixtures is augmented as the number of charges increases on each component. Although the mass spectra become increasingly complex as the number of charge states for each component increases, the individual component detection is more credible as three or more correlations occur. Thus individual components may be distinguished and their molecular weights determined although their actual identification may require additional information. The deconvolution of mixtures such as tryptic digests is circumvented when preseparation of the sample (e.g. LC/MS) is employed. Tryptic peptides tend to have only one or two ions and, in a complex mixture (>lo components), this does not provide adequate certainty for calculating the charge state (see MS/MS section). If only one charge state is present in a mass spectrum, one must resort to examining the isotopic spacing (181,calculating the spacing of logical adduct ions, or utilizing MS/MS techniques to determine the charge state as described above. However, in the case of tryptic digests, this ion is almost invariably doubly charged. For peptides that have three or greater charges a series will always be seen and a single isolated ion would not likely be observed. A final example of a typical tryptic peptide mass spectrum via electrospray ionization is one where two enzymatically cleaved peptides are cross-linked by a disulfide bridge (Figure 5, T6-Tl6, M2,from the 33.6-min retention time peak shown in Figure 2). Disulfide-bridged fragments appear as having three and four charges due to the additional basic residues and the amino termini. These mass spectra are distinguished from tryptic peptides having a histidine by the predominance of the 3+ and 4+ charge states over the doubly charged (compare Figure 5 and Figure 4A). Other more subtle but important pieces of information about the protein structure can be derived from tryptic peptide mass spectra. The carboxy terminal tryptic fragment of the protein will not contain an arginine or lysine and therefore should have a strong tendency to be singly charged. However, sufficient exceptions to this rule exist so as to exclude it from being universally applicable. In the case of met-hGH the carboxy terminal peptide T21 is disulfidebridged to T20 (Table I). The mass spectrum logically has an abundant doubly and triply charged ion correspondingto the two arginines and one amino terminus. The lack of abundant 3+ and 4+ charge states would lead one to erroneously exclude it from the class of disulfide-linked tryptic peptides typical of Figure 5. In fact, it would be difficult to distinguish it from a histidine-containing peptide as in Figure 4A without resorting to MS/MS (see below). Histidine-containing and large non-histidine-containing(-2000 u) carboxy terminal peptides will also show doubly charged ions. However, the singly charged ion will be more intense than is characteristic of a typical tryptic peptide and thus allow a tentative assignment of the carboxy terminal peptide. A last

llQ7

-1 :Son". ("I

Flgve 6. Conventionakore (4.6 mm i.d.) LC/MS TIC trace (300-2400 u) from a 2-nmol Injection of a Wyptlc digest of metMyI. The eluent ~ 8 was split postcolumn 2 0 1 such that 50 a m l n entered the 8 0 ~ and 950 NLlmin exited the split line. Unassigned peaks are due to nontryptic cleavages. For chromatographicconditions, see text.

exception to the singly charged carboxy terminal peptide mass spectrum is the case where the final carboxy terminal amino acid of the protein is an arginine or lysine. Ion Spray LC/MS with Postcolumn Splitting. Postcolumn splitting techniques can be employed with either 2.1 mm i.d. or 4.6 mm i.d. HPLC columns, which will allow for simultaneous fraction collection and on-line mass analysis. A simple, rugged postcolumn split may be formed by an empirically determined length of narrow-bore fused-silica capillary linear restridor to produce the desired split ratio. This approach allows for coupling larger diameter chromatographic columns to an ion spray or electrospray LC/MS interface. Reducing the HPLC effluent flow directed to the LC/MS interface facilitates adapting either ion spray or electrospray interfaces to higher flow HPLC systems. An example of an LC/MS analysis of the met-hGH tryptic digest with a 4.6 mm i.d. column is shown in Figure 6. Here 2 nmol of met-hGH tryptic digest were injected onto the 4.6 mm i.d. HPLC column with a total flow of 1 mL/min. Under the experimental conditions described above, 50 pL/min of the effluent was directed to the API ion source while 950 pL/min was collected (Figure 1). The differences between the corresponding data shown in Figures 2 and 6 include better sensitivity for microbore columns (70 pmol compared to 2 nmol injected) and no postcolumn splitting, but more routine, analytically rugged operation with the use of 4.6 mm i.d. columns in the latter case. The LC/MS analyses described herein were accomplished with a combination of pneumatic and electrwtatic nebulization (ion spray) as opposed to pure electrostatic nebulization (electrospray). One practical advantage of the ion spray LC/MS approach, in our hands, is the broader range available for HPLC effluent flow and solvent composition. HPLC flows from 1 to 100 pL/min may produce comparable results with the ion spray interface, while experiments with flows higher than 10 pL/min and eluents with high aqueous composition under pure electrospray conditions appear to be less rugged. Although splitting to lower flows is an alternative that can be employed with electrospray, as demonstrated above, the ability to have a broad eluent composition range (high aqueous composition at the beginning of a gradient) in which to optimize the HPLC separation provides more routine operation. Indeed, flows as high as 200 pL/min without splitting have been used for some applications of ion spray LC/MS (24b). High aqueous content HPLC mobile phases, typical of the above peptide separations, are not nebulized efficiently by only an electric field due to the high surface tension of the liquid droplets. Pneumatic nebulization eases these restrictions to produce a stable ion current signal throughout the HPLC gradient.

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b”1

1100

uB

12w

1x0

1-

1uo

IUD

E’

f

0

I450

mn

Flgm 7. Electroapray ma88 spectrum for an infusbn of a met4Gl-l tryptic digest at a flow of 2 pL/mhr. All expected tryptic pept#es are

observed.

MS/MS of Doubly Charged Peptide Molecular Ions. Sample Introduction by Infusion. Electroepray ionization mass spectra of a protein tryptic digest sample may also be obtained by simple infusion of a solution of the digest sample without preseparation of the peptide mixture through an LC column. This is practical when LC/MS experiments indicate there are no two components that yield the same mass-tocharge ratio and may be preferred over LC/MS/MS when there is limited sample available. Figure 7 shows the complex single MS mass spectrum obtained when a 50 pmol/pL solution of the met-hGH digest (Figure 2 and 6) was infused at 2 pL/min. This complex mass spectrum is composed of multiply charged molecular ions of the peptides from the digest and adducts of various components of the buffers used during the digestion. Identification of the relevant parent ions for MS/MS in mixtures of this nature benefits from a preseparation technique such as LC/MS as described above (Figures 2 and 6). Because of the numerous molecular ions with different charge states, the probability for coincidence of ions from two components at the same mass-bcharge ratio is high in tryptic digest mixtures and does occur with some of the components in this sample. A prior LC/MS experiment will indicate whether two such components are pment. Mixed daughter ion mass spectra are likely to result in these instances, which suggests the need for on-line LC/MS/MS or LC/MS/fraction collection with subsequent infusion of the purified peptides for MS/MS. However, in many instances simple infusion of the tryptic digest under MS/MS conditions can provide useful, unambiguous CID mass spectra for these peptides. Infusion of a 50 pmol/pL sample of met-hGH tryptic digest at 2 pL/min under single MS conditions revealed the molecular ions for all the tryptic fragments in this sample (Figure 7). Relative intensities of the signals for various peptides may vary by approximately a factor of 10 in the mixture with a general trend being the greater the hydrophobicity the higher the ion current signal. An example is shown (Figure 7) where T13 at mlz 693 is more abundant than T7 at m/z 762. Both peptides are singly charged and of similar mass, but yield different retention on the LC traces (Figures 2 and 6), T7 demonatrating a high degree of hydrophilicity and showing about 20% of the signal observed for T13. These determinations are more amenable to those situations where some information is known about the peptide sequences of interest. In t h w inetancea involving an unknown protein tryptic digest this technique could in principle provide helpful information to be used in concert with other available analytical biochemical data. Infusion of the digest and the sequential MS/MS technique is both time and labor efficient, requiring no sample pretreatment and approximately 1-2 min of data acquisition per

Figure 8. Daughter ion mass spectrum for met-hGH tryptic peptide T11, MW = 1361. The expected nominal masses are shown on the sequence with those In bold being observed in the mass spectrum.

peptide. To obtain these data, signal averaging of five scam over a period of 90 s was applied, resulting in the consumption of approximately 75 pmol of digest per daughter ion mass spectrum. However, this approach has two disadvantages. First, a loss of primary ion current on the molecular ions is observed to occur by a factor of approximately 10 compared to the infusion of each peptide individually. “his may be due to a competition for charge among the different components and possibly buffer suppression of ionization. Thus,if each peptide were separated by LC fraction collection an increase by a factor of 5-10 in the primary ion current can be expected. On-line LC/MS/MS would also provide an increase in ion current, but one must consider the dilution of the sample through the column and the longer analysis time. Second, infusion experiments coupled with MS/MS provide CID data on a given tryptic peptide while the others are consumed. LC/MS/MS or LC/MS/fraction collection followed by infusion circumvents this losa, and a considerable savings of the sample can result. An elegant aspect of this system lies in the amino acid sequence information available from doubly charged ions having the charge localized at both ends of the molecule. Charge-remote fragmentation (46) of internal amide bonds may explain the mass spectra that are characteristically dominated by the complementary Y/ and B, type ions (33, 37). Complications in spectral interpretation due to multiply charged daughter ions is a relatively infrequent occurrence because the charges are localized at the peptide sequence extremities. This is precisely the situation desired because it can be very difficult to determine the charge state of daughter ions, and thus their actual mass becomes ambiguous (37,391.A doubly charged daughter ion has been observed in daughter ion masa spectra of trypticlike peptides that have terminal blocking groups that are readily lost as neutral fragments and occasionally when a histidine is present in the peptide (33). The CID masa spectra for all the tryptic peptides from the infusion of the met-hGH digest show the same basic fragmentation patterns and high fragmentation efficiency with the exception of disulfide-linked peptides. The CID mass spectrum obtained from T11 (Figure 8) provides sufficient fragmentation information to allow 100% of the sequence to be determined. As the tryptic peptides increase in length above m/z 1800, however, sequence ions from the amino terminus weaken, and the disulfide-linked peptides show a more complicated mass spectrum. The regularity of the Y” and B fragmentation is interrupted, making interpretation more Wicult. Thus,for complete protein sequencing,LC/MS and MS/MS analyses of other enzymatic digests should be

ANALYTICAL CHEMISTRY, VOL. 63,NO. 13, JULY 1, 1001

t

j i

lob

575-+100,1200 75 5b 25

L

1-

.

1199

m/z 441 and 476 do not correlate with any of the predicted singly charged sequence ions and were determined to be [Ye + HI2+and [Ylo + HI2+,respectively. The appearance of these doubly charged daughter ions in the CID spectrum of a tryptic peptide indicates the possibility for the existence of an internal histidine. This speculation is supported by the coexistence of fragment ions at m/z 110 (i.e., immonium ion of histidine) and m/z 156 (Le., its corresponding Y ion). In addition, fragment ions of the type A and B were recorded up to five amino acid residues from the N-terminus. These ions facilitate the MS/MS peptide-sequencing task by offering some overlapping sequence information. On the basis of the Y ions (i-e., the C-terminus sequence information) and some of the A and B ions (i.e., representing N-terminus fragments), the sequence of this peptide may be read as V a l - V a l - A l a - G l y - V a l - A l a - ~ - ~ - n e / L e u - A . These data demonstrate the feasibility and utility for obtaining on-line LC/MS/MS analytical results for the characterization of tryptic digest composition. When preliminary LC/MS molecular weight information is combined with subsequent on-line LC/MS/MS analyses for structural characterization, one has a powerful analytical tool to use in conjunction with established, well-known biochemical techniques.

CONCLUSIONS Electrospray ionization coupled with mass spectrometry is an inherently simple technique to implement with samples dissolved in aqueous solvents. It has a basic requirement that the sample to be analyzed must be dissolved and either be ionized in solution or associated with an ion during the formation of the gas-phase ion before the ionization/desorption procees can occur. The chemical environment in the solution should be suitable for assisting the ion evaporation process. This usually means that the pH of the solution should be adjusted to ionize the sample without an excessive increase in the ionic strength of the solution. Desorption of condensed-phase ions into the gas phase also occurs most efficiently at atmospheric pressure where solvent evaporation rates are the highest. The latter pertains because the heat of vaporization may be supplied more readily at atmospheric pressure than under high-vacuum conditions. These combined reasons make the ion evaporation mechanism an excellent candidate for coupling to HPLC or other condensed-phase separation techniques (47). The two common LC/MS problems of forming gas-phase ions from materials dissolved in HPLC eluents and the adaptation of the mass spectrometer to accommodate liquids in a high-vacuum system are simultaneously solved. The dual characteristics of mild ionization and the production of multiply charged ions allow the system to be operated effectively for on-line LC/MS under HPLC conditions commonly used for proteins and peptides. Pneumatic nebulization of the electrosprayed liquid adds operation simplicitymaking the technique readily compatible with conventional HPLC separation modes for peptides requiring high aqueous content gradients and TFA buffers. Complex sample mixtures such as the tryptic digests described in this work may be analyzed by at least three different but related approaches. The first and perhaps simplest is the direct "infusion" of a solution containing, for example, 50 pmol/pL of a crude tryptic digest at a flow of 2 pl/min through the ion spray LC/MS interface in the single MS mode. In this experiment a slow scan rate (25s from m / z 300 to 2400) with a mass step of 0.1 u/sample provides mass assignment to the nearest dalton and a complex mass spectrum showing ions for all the detected peptides. In principle, the molecular weight for each peptide may be determined to within 1 without any chromatographic separation. Once the precursor ions of interest have been determined, the same infusion experiment may be repeated under CID conditions

1200

ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991

and the corresponding CID mass spectrum obtained. This also precludes chromatographic separation. However, the chemical complexity of these samples may cause reduced response to certain components and ambiguities may result due to interferences from dissolved salts or other components. In contrast, on-line LC/MS and LC/MS/MS experiments “clean up” the sample on-line and provide less ambiguous results, which may result from interfering components. However, the maas spectrometer must be scanned considerably faster (3-5 slscan) to keep up with the chromatographic time scale, so a mass peak sampling rate of 0.5 u is required for adequate mass detection and assignment. As the mass range scanned increases this imposes added burdens on mass spectrometer data systems. The system described is adaptable to HPLC column diameters ranging from packed capillaries (48) to 4.6 mm i.d. columns with appropriate splitting such that the total quantity entering the ion source may range from 1to 100 pL/min for optimal ion spray LC/MS operation. Split ratios as high as 95% and aqueous compositions approaching 100% can be utilized, allowing recovery of the majority of the sample for use with alternative techniques. The utility of this capability in the peptide field, in addition to real-time separation/mass determination of peptidelprotein mixtures, is simultaneous fraction collection for sequencing by classical techniques or MS/MS. On-line ion spray LC/MS is applicable to many types of protein digests. Recent data have shown that glycopeptides from digests of glycoproteins may be similarly determined by this approach and offer possibilities for mapping posttranslationally modified proteins (49,50). Tryptic peptides represent a special case where the majority of the ion current resides in their doubly charged molecular ions. MS/MS characterization of the doubly charged molecular ions of these peptides reveals a high degree of predictability with respect to fragmentation patterns, thus making their CID mass spectral interpretation and the determination of their sequence information an easier task. On-line LC/MS/MS analyses of protein tryptic digests provides an elegant means of obtaining the optimum amount of primary structure information with a minimum consumption of sample or time.

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RECEIVED for review September 4,1990. Accepted March 28, 1991. E.C.H. and J.D.H. gratefully acknowledge the Eastman Kodak Company and Sciex for partial financial support of this work.