A Mass Spectrometric Method for Carbohydrates and Peptides

Aug 10, 2005 - Prozyme (San Leandro, CA) and used without further purification. ... trap mass spectrometer (San Jose, CA) in triplicate. Tandem mass...
0 downloads 0 Views 230KB Size
Download PDF
Anal. Chem. 2005, 77, 5886-5893

STEP (Statistical Test of Equivalent Pathways) Analysis: A Mass Spectrometric Method for Carbohydrates and Peptides Mary L. Bandu,† Jonathan Wilson,‡ Richard W. Vachet,‡ Dilusha S. Dalpathado,† and Heather Desaire*,†

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, and Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003

We have recently developed a new mass spectrometry method, the STEP (statistical test of equivalent pathways) analysis that uses ion abundances in two tandem mass spectrometry experiments to obtain genealogy information about product ions present in mass spectra. The method requires minimal sample, and it can be performed using a conventional quadrupole ion trap mass spectrometer. To obtain genealogy information, STEP ratios are calculated by comparing the relative abundances of product ions in two MS/MS experiments. These ratios are directly related to the origin of the product ions. Product ions that result directly from the precursor ion always have STEP ratios that are e1. Ions that result from secondary fragmentation pathways have STEP ratios that are significantly larger than the primary ions, based on a Q test of statistical significance. Consequently, the type (primary or secondary) of all the product ions in an MS/MS experiment can easily be identified in this analysis. The STEP method is applied herein to peptides and carbohydrates, and the STEP results are consistent with validation data for 95% of the ions in this study. This new method has many applications in carbohydrate and peptide analysis. It can be used to support mechanistic studies of peptide fragmentation, and it is useful for discriminating among various isomeric carbohydrates, without the need for reference standards. Several examples are presented to demonstrate the reliability of this method, and an example showing how the method benefits carbohydrate sequencing is also provided. Mass spectrometry (MS) has become one of the premier techniques for analysis of proteins and carbohydrates due to the development of the ionization methods electrospray ionization and matrix-assisted laser desorption/ionization, which can ionize biological molecules in a nondestructive fashion.1-7 In addition, * To whom correspondence should be addressed: (e-mail) [email protected]; (tel) (785) 864-3015. † University of Kansas. ‡ University of Massachusetts. (1) Anderson, J. S.; Svensson, B.; Roepstorff, P. Nat. Biotechnol. 1996, 14, 449457. (2) Biemann, K. Annu. Rev. Biochem. 1992, 61, 977-1010. (3) Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology; MCC Press: San Diego, 2003.

5886 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

tandem MS (MS/MS) experiments extend mass spectrometry’s analytical capabilities by providing structural information about these biomolecules. MS/MS analysis of peptides results in product ion spectra that can be used to identify amino acid content and sequence information.1-2,8,9 MS/MS of carbohydrates also has the potential to provide sequence information for glycans.7,10 However, since carbohydrate structures are not linear but highly branched, collision-induced dissociation (CID) spectra are more difficult to interpret. Carbohydrate sequence analysis typically involves several experimental approaches, not just MS/MS.6 Analysis of peptides and carbohydrates for sequence information using MS is dependent on extensive dissociation of precursor ions in CID experiments, to produce spectra containing numerous product ion peaks.7,11 To obtain extensive fragmentation, enough energy must be input so that multiple fragmentation channels are energetically accessible.12 This can promote the formation of secondary fragment ions. Secondary fragmentation is the result of continued dissociation of primary fragments due to excess internal energy (precursor ion f primary product ion f secondary product ion).13,14 Secondary ions contribute to informationrich spectra by providing many more product ion peaks and, therefore, additional information about amino acid sequence and carbohydrate linkages. Identifying product ions that are the result of primary or secondary fragmentation is important for sequence analyses and mechanistic studies of peptide fragmentation. The mechanism describing peptide fragmentation that occurs at acidic residues was supported by the fact that backbone cleavages on the C-terminal side of acidic residues generated the primary (4) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (5) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (6) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-369. (7) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (8) Papaynnopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (9) Wysocki, V. H.; Resing, K. A.; Zhang, Q.; Cheng, G. Methods 2005, 35, 211-222. (10) Gaucher, S. P.; Morrow, J.; Leary, J. A. Anal. Chem. 2000, 72, 2331-2336. (11) Steen, H.; Mann, M. Nat. Rev. 2004, 5, 600-711. (12) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry; VCH Publishers: New York, 1988. (13) Vekey, K. J. Mass Spectrom. 1996, 31, 445-463. (14) Vachet, R. W.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1996, 7, 11941202. 10.1021/ac050722e CCC: $30.25

© 2005 American Chemical Society Published on Web 08/10/2005

products.15-18 Because primary, lower series y- and b-ions form at acidic residues, identifying these ions as “primary” could be used to identify the site of acidic residues, since lower series yand b-ions frequently result from secondary fragmentation.19,20 For carbohydrates, identifying secondary fragmentation has even greater advantages. Carbohydrates are branched species that undergo extensive fragmentation at their glycosidic bonds during CID.21 The ability to determine the carbohydrate structure is complicated after secondary fragmentation, because one cannot assume that a neutral loss came from one specific location on the structure; it could have come from a combination of two bonds cleaved, from two different carbohydrate branches.21 This ambiguity can lead to ambiguous structural assignments. By identifying the product ions that come from breaking only one glycosidic bond, structural assignments will be less ambiguous.7,21 All the monosaccharides lost in a given primary fragment must originate from one branch of the carbohydrate. Clearly, the identification of secondary fragment ions in tandem mass spectra is important in both carbohydrate and peptide analysis. Currently, two validated mass spectrometric methods are employed for the identification of secondary fragments in MS/ MS data: double-resonance experiments20,22,23 and the construction of breakdown curves.24-26 Double-resonance experiments are achieved by performing CID experiments while simultaneously ejecting a product ion present in the spectrum. By ejecting this product ion, all secondary product ions that originate from the ejected ion will be eliminated in the final tandem mass spectrum. The construction of breakdown curves, which graph ion abundance versus collision energy or activation time, also provides information about secondary fragmentation. Unfortunately, neither of these established techniques is readily amenable to routine peptide and carbohydrate analysis if sample sizes are very limited. Double-resonance experiments require customized modification to instruments,22 trained operators, and relatively large quantities of sample, so adequate control experiments can be performed. Similarly, the construction of breakdown curves requires the collection of numerous spectra at multiple collision energies or activation times,24-26 and the application of breakdown curves for determining secondary fragmentation has only been achieved in mass spectrometers with quadrupole or hexapole collision cells, not quadrupole ion traps. While these methods provide powerful tools for structural analysis, their applicability is limited because (15) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154. (16) Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 58045813. (17) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406. (18) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2000, 35, 1382-1390. (19) Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1996, 7, 233-242. (20) Vachet, R. W.; Ray, K. L.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1998, 9, 341-344. (21) Harvey, D. J. J. Am. Soc. Mass. Spectrom. 2000, 11, 900-915. (22) Vachet, R. W.; McElvany, S. W. J. Am. Soc. Mass Spectrom. 1999, 10, 355359. (23) Colorado, A.; Shen, J. X.; Vartanian, V. H.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (24) Speir, J. P.; Amster, I. J. J. Am. Soc. Mass Spectrom. 1995, 6, 1069-1078. (25) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 1998, 33, 705-712. (26) Crowe, M. C.; Brodbelt, J. S.; Goolsby, B. J.; Hergenrother, P. J. Am. Soc. Mass Spectrom. 2002, 13, 630-649.

of the large sample requirements and, in the case of doubleresonance experiments, customized instrumentation. This study introduces a truly unique statistical method to identify secondary fragmentation in tandem mass spectra, by performing just two MS/MS experiments on a conventional quadrupole ion trap mass spectrometer. This new approach facilitates sequencing of carbohydrates, and it could be used to facilitate peptide analysis as well. The STEP (statistical test of equivalent pathways) analysis utilizes ion abundances from highand low-dissociation experiments. From these abundances, STEP ratios are calculated. All primary product ions have statistically similar STEP ratios, so a simple Q test identifies fragments that are statistically different and therefore due to secondary fragmentation. This method requires minimal sample and minimal analysis, and it is accomplished on an (unmodified) quadrupole ion trap mass spectrometer. A validation of the method is presented, followed by a demonstration of how the STEP method facilitates structural identification of carbohydrates. METHODS Sample Preparation. All peptides used in this study were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved and diluted in 0.5% ammonia/methanol to a final concentration of 1 × 10-4 M. (This concentration is well above the level required, but no attempt was made to determine the detection limit using the STEP method.) The carbohydrate M3N2 was purchased from Prozyme (San Leandro, CA) and used without further purification. Procainamide-derivatized maltopentaose was synthesized using a method described previously.27 Carbohydrates were diluted in a 50:50 methanol/water solution and further diluted to 1 × 10-5 M in HPLC grade methanol (Fisher Scientific, Pittsburgh, PA). Acquiring Tandem Mass Spectra. The MS/MS data used for STEP analysis were acquired on a ThermoFinnigan LCQ ion trap mass spectrometer (San Jose, CA) in triplicate. Tandem mass spectra were collected in positive ion mode using the standard, “profile” mode peak feature. The sample was directly infused from a syringe pump at a flow rate of 5-7 µL/min. Tuning was performed to optimize signal intensity for singly charged ions. These precursor ions were isolated using an isolation width of 2-4 Da. For each MS/MS experiment, 100 scans (each containing 3 “microscans”) were collected and averaged. An activation time of 30 ms and a qz value of 0.25 were used for all MS/MS experiments. High-dissociation MS/MS is defined as the energy needed to deplete the precursor ion to 0-2% relative abundance. From this spectrum, the most abundant product ion is identified and used to determine the low-energy regime. Low-dissociation MS/MS is defined as the energy needed to produce a spectrum where the most abundant ion in the high-dissociation spectrum has a relative abundance of ∼30% in the low-dissociation spectrum. Calculating the STEP Ratio. From the spectrum list, the product ions above 2% relative abundance in the high-dissociation spectrum were identified and utilized for the study. Isotope peaks were excluded and not considered as product ions. The total product ion area was calculated by summing all relative abundance values from the lowest m/z value up to the precursor value minus 5. (27) Dalpathado, D. S.; Jiang, H.; Kater, M. A.; Desaire, H. Anal. Bioanal. Chem. 2005, 381, 1130-1137.

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5887

precursor - 5

total product ion area )

∑

m/z abundance

(1)

0

For example, the precursor ion for the derivatized maltopentaose sample is m/z 1048. The total product ion abundance is equal to the sum of all relative abundances up to m/z 1043. This area is the total product ion area, and it excludes the area of the precursor ion. Calculating values for a specific product ion’s area is accomplished by summing the relative abundance values from product ion m/z, (0.5 unit. +0.5

product ion area )

∑ m/z product ion

(2)

-0.5

For example, the product ion area for an ion at m/z 702.5 would be calculated by summing the abundance values from m/z 702.0 to 703.0. A ratio for each product ion was calculated by eq 3. This

ratio of product ion area ) product ion area/total product ion area (3) ratio is acquired for each product ion in the low- and highdissociation spectra that is above 2% relative abundance in the high-dissociation spectrum. The STEP ratios are then calculated via eq 4.

STEP ratio ) high-dissociation ratio of product ion area/ low-dissociation ratio of product ion area (4) To determine whether a product ion is secondary, a Q test is employed. STEP ratios of e1 are always assigned as primary fragments. Using these primary fragments, the Q test is performed to determine whether the other fragments having values over 1 are significantly different. The 90% confidence interval is used in all the analyses herein.28 All samples were analyzed in triplicate. Double-Resonance Experiments. A Bruker Esquire∼LC (Bruker Daltonics Inc., Billerica, MA) quadrupole ion trap mass spectrometer was used for the double-resonance experiments. The double-resonance waveforms were output from a Wavetek model 39 arbitrary waveform generator (Fluke Corp., Everett, WA). The appropriate timing of the waveform from the arbitrary waveform generator was provided by a model DG535 digital delay pulse generator (Stanford Research Systems, Inc., Sunnyvale, CA). During the double-resonance experiments, the precursor ion of interest was isolated and subjected to resonance excitation using the Esquire∼LC hardware. Simultaneous to the application of this resonance excitation signal, another signal of the appropriate frequency supplied by the arbitrary waveform generator was applied to the entrance end cap to resonantly eject the product ion of interest as it was formed. Two types of control experiments were performed for each double-resonance experiment to verify the accuracy of the results. In the first control, the product ion of interest was generated, isolated, stored at the same qz value as it was during double(28) Rorabacher, D. B. Anal. Chem. 1991, 63, 139-146.

5888

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

resonance experiment, and subjected to the same resonance ejection signal as during the double-resonance experiment. The purpose of this control experiment was to ensure complete ejection of the product ion. The second control experiment was similar to the first except that secondary products ions of interest were generated, isolated, stored at the same qz value as during the double-resonance experiment, and subjected to the same resonance ejection signal as during the double-resonance experiment. This control experiment was designed to confirm that the doubleresonance waveform did not reduce the peak intensities of these secondary product ions via off-resonance dissociation. RESULTS AND DISCUSSION Theory and Methodology for the STEP Analysis. Breakdown curves can provide information about primary and secondary product ions in MS/MS data.12,25 The STEP analysis uses the same theoretical basis of these original findings; at increasing collision energy, secondary product ions become more abundant and primary product ions are depleted. While the theoretical basis is unchanged, the methodology of the STEP analysis has two significant modifications: (1) Only two tandem mass spectra are required, and (2) the STEP analysis is performed using a quadrupole ion trap mass spectrometer instead of a quadrupolebased mass spectrometer, which is typically utilized for the identification of secondary fragmentation using breakdown curves. Traditional breakdown curves used to identify primary and secondary product ions are not obtainable in conventional ion trap instruments because high-energy CID data, where primary product ions become depleted substantially, is not achievable. As a result of this energy constraint, CID spectra acquired on conventional ion trap instruments indicate that virtually all product ion abundances increase with increasing activation, as the precursor ion becomes depleted. For example, see ref 23. These breakdown curves are not used to distinguish primary and secondary product ions in the CID spectra because all the ion abundances are increasing. The STEP method can distinguish primary and secondary fragmentation in a quadrupole ion trap mass spectrometer by tracking product ion abundances using a different approach. Traditional breakdown curves typically plot “relative abundances,” on the y axis.12 For STEP analysis, the analogous parameter is a quotient: product ion abundance, divided by the sum of all the product ions (which excludes the precursor ion). With this modification, primary product ion percent areas do not increase significantly with increasing collision energy, while secondary product ion areas, expressed as a percent of all product ions, increase substantially with increasing activation voltage. Figure 1 shows how expressing product ion abundance, divided by the sum of all product ions, can be used to generate modified breakdown curves in an ion trap mass spectrometer. Figure 1A contains primary product ions in the tandem MS spectrum of a derivatized carbohydrate. These ions were validated as primary fragments using double-resonance experiments, and the validation is discussed in the next section. In Figure 1A, all primary product ion percents decrease or remain constant with increases in activation voltage. This result is expected due to the production of secondary fragments from primary ions. Nonequivalent pathways that result in secondary fragmentation will increase

Figure 1. Modified breakdown curve for product ions of a derivatized carbohydrate sample (DEAEAB-derivatized maltopentaose.) (A) Primary product ions. (B) Secondary product ions. The two sets of ions are readily distinguished from each other. Arrows on the graphs indicate the activation amplitude used in the STEP analysis.

in percent product ion abundance with increasing activation voltage. Figure 1B demonstrates this trend. The STEP method utilizes only one high-dissociation and one low-dissociation spectrum to determine whether product ion areas are increasing or decreasing. As shown in Figure 1, the red arrows indicate the collision energy utilized for high-dissociation and lowdissociation data acquisition. By comparing ion abundance areas at these two collision energies and calculating a ratio, primary fragments are identified by decreases in product ion area and a ratio of e1. From these values for primary fragmentation, the STEP ratios for other product ions are evaluated using the Q test. Those that are statistically similar to primary ions are identified as primary. Those that are significantly different (at the 90% confidence interval) are due to secondary fragmentation. STEP Ratios for Identification of Secondary Dissociation in Peptides. Two peptides are shown in Figure 2, with peptide fragmentation nomenclature labeled according to Biemann and Roepstorff notation.29,30 The calculated STEP values are indicated (29) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (30) Roepstorff, P. Biomed. Mass Spectrom. 1984, 11, 601.

in parentheses, and the corresponding classification determined by double-resonance experiments is shown (1° ) primary product, 2° ) secondary product). When the STEP ratios for each of the product ions are compared to double-resonance data, the ions with the largest STEP ratios are always due to secondary fragmentation. For example, in Figure 2A, the a4 ion (m/z 397) for Leu-Enk has three sources. Approximately 11% of this ion is the result of primary fragmentation from the parent ion, 2% from secondary fragmentation of the b5 ion, and 87% from secondary fragmentation of the b4 ion.20 Because this ion is predominantly the result of secondary fragmentation (89% secondary), the STEP ratio is large, ∼2.2. The only ion with a larger STEP ratio is m/z 278, the b3 ion. Doubleresonance data also indicate this ion is a secondary product (see Supporting Information). It primarily originates from the b4 ion. All the ions with low STEP ratios (1.4 or less) are all primary ions. For example, double-resonance experiments indicate that the y2 ion, m/z 279, originates directly from the parent ion, not through the y3 ion. Likewise, the ions m/z 425, and 336 also are primary ions, as they are the b- and y-ions with the highest m/z Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5889

Figure 2. MS/MS data for two peptides. Each ion greater than 2% relative abundance is labeled with the calculated STEP ratio (in parentheses). The origin of the ions (primary or secondary) was determined independently using double-resonance experiments.

values observed in the spectrum. In addition, the ion m/z 538 is a loss of H2O, which makes it a primary ion; it has the lowest STEP ratio of all the product ions. Figure 2B is the tandem MS spectrum of Val-Glu-Pro-Ile-ProTyr (VEPIPY). The same trend, that secondary products always have higher STEP ratios than primary product ions, is also observed. The product ions at m/z 411 (a4) and 326 (b3) consistently have the largest STEP ratios compared to all other product ions in the spectrum, and both these ions are due to secondary fragmentation. This is validated through doubleresonance experiments that indicate these fragments both originate from m/z 439 (b4). The a4 ion (m/z 411) is ∼100% from b4, and b3 (m/z 326) is ∼80% from b4. The ion with the third-highest STEP value is m/z 279. The double-resonance data indicate m/z 279 is depleted by ∼50% when m/z 489 is resonantly ejected during CID experiments. Since this ion is partially due to primary fragmentation and partially due to secondary fragmentation, a 5890

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

STEP ratio between the STEP ratios for the primary and secondary ions is reasonable. The STEP method assigns the b4 and y4 ions of VEPIPY as 1° because their STEP ratios are very low (less than 1). The assignment, that these ions are primary, is consistent with the mass spectral data, since they are the b- and y-ions with the largest m/z in the spectrum. In other words, larger b- and y-ions from which they could arise are not observed, so they most likely originate from the precursor ion. STEP Ratios for Secondary Dissociation in Carbohydrates. The derivatized maltopentaose sample in Figure 3A was subjected to STEP analysis, and the STEP results were validated by double-resonance experiments (in Supporting Information). For this sample, double-resonance experiments confirm that all the glycosidic cleavage ions, m/z 886, 724, 562, and 400, originate directly from the precursor ion. The product ion, m/z 975, is also a primary product ion as it is the product ion in the spectrum

Figure 3. MS/MS data for two carbohydrates. Circles represent hexoses; squares represent N-acetylhexosamines. In (A), the carbohydrate is derivatized via reductive amination. Each ion greater than 2% relative abundance is labeled with the calculated STEP ratio (in parentheses). The origin of the ions (primary or secondary) was determined through independent validation.

with the largest m/z. It corresponds to loss of diethylamine. The ions with the largest STEP ratios, 813, 651, 489, and 327, are all due to a combination of glycosidic cleavage and loss of diethylamine. Since the monosaccharides and the diethylamine are at two different ends of the molecule, these ions must be the result of secondary fragmentation. This assertion is supported by doubleresonance experiments that indicate ∼100% of m/z 489 originates from a secondary fragmentation pathway. Its precursors are m/z 562 and 975. The remaining two ions in this spectrum are m/z 608 and 446. The STEP ratios for these ions are higher than all the confirmed primary ions but lower than all the ions that are confirmed to be secondary products. These ions most likely originate from a rearrangement involving both ends of the molecule because they are not consistent with any known carbohydrate fragmentation. However, no double-resonance experiments identified them as secondary fragments, so these ions cannot be validated as primary or secondary. The dissociation of the second carbohydrate in this study, Hex3HexNAc2, can be assigned without double-resonance experiments, because of the unique structure of this carbohydrate. (Hex ) hexose, HexNAc ) N-acetylhexosamine.) The Hex’s and HexNAc’s are at two different ends of the molecule, so any neutral loss containing both Hex and HexNAc must result from secondary

fragmentation. In addition, because the hexoses are branched, loss of two hexoses must also result from breaking two bonds. For this carbohydrate, there are only three primary ions in the mass spectrum, loss of water (m/z 893), and m/z 749 and 690, which correspond to loss of one Hex or one HexNAc, respectively. All other ions in the spectrum are secondary fragments of these primary ions. For example, m/z 587 is a loss of one hexose from m/z 749, and m/z 731 is a loss of water from m/z 749 (and/or a loss of a hexose from m/z 893). Finally, m/z 528 and 366 are both fragments of m/z 690; they result from loss of one or two hexoses from this ion. The ion at m/z 425 can originate from primary, secondary, or tertiary dissociation, so it most likely contains at least some secondary character. It should be noted that m/z 366 can only be formed by cleaving three bonds. As expected, the STEP ratios for all the primary ions are always lower than the STEP ratios for all the secondary ions. In addition, the ion with the highest STEP ratio, m/z 366, can only be formed when three glycosidic bonds are cleaved. Where To Draw the Line (Distinguishing Primary from Secondary). The data clearly demonstrate that secondary product ions always have larger STEP ratios than primary product ions; however, using STEP analysis to determine primary and secondary fragments requires that one know which STEP ratios are large enough to allow identification of secondary fragments and which STEP ratios identify primary fragments. We have empirically determined that the best method to identify the “cutoff” between primary and secondary STEP ratios is use of a Q test. The Q test addresses the question of whether one number is significantly different from a given group of numbers. In STEP analysis, any ion with a STEP ratio of less than 1 must be a primary ion, because its intensity is decreasing relative to other ions, as the activation is increased. By using all of the ions that have a STEP ratio of less than 1 as the “control” group, any ion with a STEP value of greater than 1 can be subjected to the Q test, to determine whether it is statistically different from the control group, and therefore a secondary ion, or statistically similar, and therefore a primary ion. Using the Q test to determine primary and secondary ions is more effective than simply setting a rigid cutoff point for distinguishing the two sets of ions, and this is likely due to the fact that when the Q test is used, the cutoff that separates primary and secondary varies, depending on the STEP ratios of the ions in the control group. For example, the cutoff values that separate primary and secondary ions for VEPIPY, the first compound in Table 1, are slightly different for the three replicates of this compound. This is because the STEP ratios for the primary ions in the “control groups” were all slightly different. In the first trial for VEPIPY, the two ions in the control group had STEP ratios of 0.79 and 0.83. When these STEP ratios are used, the cutoff between primary and secondary is 1.46, based on the Q test. For the second and third trials, the cutoff between primary and secondary is at a STEP value of 1.67 and 1.33, respectively. These cutoff values are depicted in the solid lines that separate the primary and secondary ions in Table 1. Using this Q test at the 90% confidence interval, assignments of primary or secondary were made for all the product ions in this study. See Table 1. When these results are compared to the validation data, all of the ions that were validated as being Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5891

Table 1. STEP Analysis versus Validation Data for Assigning Secondary Fragmentation

secondary product ions were also assigned as secondary product ions using the Q test. Almost all of the ions that were validated to be primary product ions were identified as primary products, using the Q test. Of the 93 product ions in this study, only 5 were misidentified, using the Q test (95% were correctly identified). This seems reasonable, since the Q test, set at the 90% confidence interval, identifies a 90% chance that the ions falling outside the range are from significantly different pathways. With this in mind, it is unreasonable to expect to get 100% of the identifications correct. 5892

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

If one wanted to be certain that no secondary ions were misidentified as primary, a higher confidence interval could be used (like the 98% CI). Likewise, if the goal was to be certain that no primary ions were misidentified as secondary, a lower confidence interval could be used. The 90% CI was chosen because it appears to provide the lowest number of misidentifications, overall. Application to Carbohydrate Analysis. The spectrum in Figure 4A shows MS/MS data for an N-linked carbohydrate, and the STEP ratios for each of the product ions are above the ions. The ions with STEP ratios less than 1 were used to determine

analysis, complete sequence and branching information can be inferred for a wide variety of carbohydrates. A report detailing the implementation of the STEP method into a general strategy for carbohydrate structural analysis will be presented separately. Additional developments for STEP analysis that are currently under investigation include the following: application of the method to doubly charged species; determining whether secondary fragmentation could be distinguished from tertiary fragmentation; application of the method to CID data from triple quadrupole mass spectrometers and linear ion traps; and implementing the method in LC-MS/MS analyses.

Figure 4. (A) MS/MS data for a biologically relevant carbohydrate. Each ion greater than 2% relative abundance is labeled. STEP ratios are on the spectrum along with the origins of the ions (primary or secondary) as identified by STEP analysis. (Circles represent hexoses; squares represent N-acetylhexosamines.) (B) Six biologically relevant, isomeric carbohydrates. Only one structure, 4-1, is consistent with results of the STEP analysis in (A), so the others could be ruled out as possibilities, if the structure of the carbohydrate were unknown.

which ions were significantly different, at the 90% confidence interval. In this example, the Q test indicates that the cutoff between primary and secondary ions occurs at a STEP ratio of 3.34, so m/z 1276, with a STEP ratio of 1.65, is clearly a primary ion, and m/z 1055, with a STEP ratio of 3.35, is a secondary ion. The STEP assignments (primary or secondary) are labeled on the spectrum. This information could be used to infer the structure of the carbohydrate, if it had been unknown. In this analysis, m/z 1055 (loss of HexNAc2Hex) is produced through secondary fragmentation, while m/z 1276 (loss of HexHexNAc) and 1114 (loss of Hex2HexNAc) both originate directly from the precursor ion. This information provides considerable information about the carbohydrate. The correct structure must contain a (HexHexNAc) disaccharide and a trisaccharide that has a composition of (Hex2HexNAc). Both these fragments must be cleavable from the rest of the carbohydrate by breaking just one bond each. This information allows one to rule out many biologically possible but incorrect structures for this carbohydrate. In Figure 4B, six isomeric, biologically relevant carbohydrate structures are depicted. Only one of them, (4-1), has a structure consistent with the STEP data. All the other five structures must produce either m/z 1276 or 1114 through secondary fragmentation. Since STEP analysis assigns these ions as primary, all the structures except 4-1 can be ruled out as possible structure candidates. By combining STEP analysis with current MS methods of carbohydrate

CONCLUSION This study provides a validated method to quickly obtain primary and secondary fragmentation information from MS/MS data for peptides and carbohydrates using a conventional quadrupole ion trap mass spectrometer. The method utilizes ion abundances from a high- and a low-dissociation spectrum list to calculate a STEP ratio. Product ions that have STEP ratios less than 1 are immediately identified as primary ions, and they are used to determine whether other ion ratios are significantly different, based on the Q test. Secondary fragments have STEP ratios that are significantly different from primary ratios, so they can be correctly identified as originating from some source other than the precursor ion in MS/MS data. The method only requires acquisition of two mass spectra, and it can be completed with limited sample consumption on a commercial instrument. Identifying primary and secondary product ions greatly facilitates carbohydrate structural analysis, as demonstrated herein, and it can also be used in mechanistic and sequencing studies of peptide fragmentation. This method was specifically designed for carbohydrates and peptides. The polymeric nature of these species likely contributes to the overall success of this analysis, because several bond cleavages of the same type are observed. Ongoing studies in STEP analysis are focusing on how the type of bond cleavage affects the STEP ratio. A potential limitation of the method may be that it is not readily applicable to nonpolymeric species. The utility of the STEP analysis for singly charged peptides and carbohydrates is presented here, and additional studies that further explore the scope and limitations of the analysis are currently under investigation. ACKNOWLEDGMENT Funding from this project was provided by the National Institutes of Health, projects 1 P20 RR17708-01 and R01GM077226. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 27, 2005. Accepted July 6, 2005. AC050722E

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5893