Anal. Chem. 2001, 73, 1733-1739
On-Chip Proteolytic Digestion and Analysis Using “Wrong-Way-Round” Electrospray Time-of-Flight Mass Spectrometry Iulia M. Lazar,† Roswitha S. Ramsey, and J. Michael Ramsey*
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365
Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using “wrong-wayround” electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/µL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique. Mass spectrometry (MS) has emerged today as an indispensable tool in protein analysis. Strategies for identifying proteins include peptide mapping where the mass-to-charge ratios of peptide fragments produced by site-specific enzymes are measured and correlated with information contained in protein databases. In the peptide sequence tag approach, partial amino acid sequence data from tandem MS analysis, together with the residual mass of the peptide and N- and C-terminal segments of the peptide, are used to identify the protein from various databases. Typically for mixtures, proteins are separated using one- or two-dimensional gel electrophoresis and detected using an appropriate technique (e.g., Coomassie Blue or silver staining). The isolated bands or “spots” are then enzymatically digested either directly in the gel or following transfer onto a membrane. The resultant peptides are extracted, desalted, preconcentrated, and further analyzed, frequently by liquid chromatography (LC)-MS or LC-MS/ MS.1-10 * To whom correspondence should be addressed: (phone) (423) 574-5662; (fax) (423) 574-8363. † Present address: The Barnett Institute/Northeastern University, Boston MA 02115-500. (1) Huang, E. C.; Henion, J. D. J. Am. Soc. Mass Spectrom. 1990, 1, 158-165. (2) Takada, Y.; Nakayama, K.; Yoshida, M.; Sakairi, M. Rapid Commun. Mass Spectrom. 1994, 8, 695-697. 10.1021/ac001420+ CCC: $20.00 Published on Web 03/20/2001
© 2001 American Chemical Society
On-line peptide separation prior to MS detection has several demonstrated benefits: (1) removal of matrix components, including salts and detergents in the sample, (2) preconcentration of the fragments at the head of the separation column, and (3) isolation of the mixture components. All of these improve the signal-to-noise ratios and result in simpler, easier to interpret mass spectra. Effecting a separation, however, is time-consuming and limits overall sample throughput. Sample loss or contamination due to adsorption on the column packing material may also be significant, reducing sequence coverage for the protein. Furthermore, MS/MS analysis must be performed within the finite time frame associated with the elution of a given peak. Nanoelectrospray11-16 has been found useful for the sensitive analysis of complex peptide mixtures obtained from proteolytic digestions, often eliminating the necessity of performing prior chromatographic separations.13-18 It has been used with MS/MS in the peptide sequence tag method, for instance, to identify lownanogram quantities of proteins isolated from one-dimensional gels.12,14,16 The benefits associated with the technique include high sensitivity, low analyte consumption, compatibility with a wide range of sample matrix or buffer compositions, and overall simplicity. Nanoelectrospray also extends the time available for MS analysis of a given sample and improves the quality of the spectra due to increased signal summing and/or averaging (3) Davis, M. T.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1997, 8, 1059-1069. (4) Le, J. C.; Hui, J.; Haniu, M.; Katta, V.; Rohde, M. F. J. Am. Soc. Mass Spectrom. 1997, 8, 703-712. (5) Wu, J.-T.; Huang, P.; Li, M. X.; Lubman, D. M. Anal. Chem. 1997, 69, 29082913. (6) Li, M. X.; Wu, J.-T.; Parus, S.; Lubman, D. M. J. Am. Soc. Mass Spectrom. 1998, 9, 701-709. (7) Chen, Y.; Wall, D.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1998, 12, 1994-2003. (8) Cao, P.; Moini, M. Rapid Commun. Mass Spectrom. 1998, 12, 864-870. (9) Davis, M. T.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1998, 9, 194-201. (10) Raida, M.; Schulz-Knappe, P.; Heine, G.; Forssmann, W. G. J. Am. Soc. Mass Spectrom. 1999, 10, 45-54. (11) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (12) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (13) Wilm, M. S.; Mann, M. Anal. Chem. 1996, 68, 1-8. (14) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-467. (15) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527-533. (16) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (17) Fligge, T. A.; Kast, J.; Bruns, K.; Przybylski, M. J. Am. Soc. Mass Spectrom. 1999, 10, 112-118. (18) Blackburn R. K.; Anderegg, R. J. J. Am. Soc. Mass Spectrom. 1997, 8, 483494.
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1733
capabilities. Recent developments in time-of-flight (TOF) and hybrid quadrupole/TOF instrumentation that provide resolution in excess of 10 000 and mass accuracy of less than 10-30 ppm significantly increase the specificity of database searches, making direct nanoelectrospray TOFMS analysis an attractive alternative to HPLC-MS methods for peptide mapping. One disadvantage associated with conventional nanoelectrospray MS, however, is that the technique is not directly amenable to high-throughput analysis. Combining nanoelectrospray with microfabricated fluidic devices, however, may overcome this limitation. In the past few years, devices that enable complete sample processing on miniaturized platforms have been developed.19-22 A variety of operations, such as preconcentration, separation, analyte derivatization or chemical modification, and detection have been demonstrated on these compact, relatively simple “labon-a-chip” devices.19-22 Several research groups have also interfaced microchips with MS using electrospray ionization (ESI),23-35 and microfabricated devices with multiple parallel sample delivery channels for high-throughput ESI analysis have been reported.24,26,29,36 We recently developed a microchip nanoelectrospray source capable of providing subattomole sensitivity for peptide and protein samples.35 Fluid is delivered as a result of electrostatic forces and capillary action, as in conventional nanoelectrospray sources, eliminating the need for external devices (detached electroosmotic pumps, pressure or vacuum sources) to promote fluid flow through the microchip to the tip. Here we report on its applicability for the rapid analysis of tryptic digests using TOFMS detection. Reaction conditions that accelerate the digestion process in solution and allow sensitive, direct MS analysis of the digestion mixture, without any intermediate cleanup or solvent exchange, were determined. Results obtained using basic pH digestion conditions, favorable for enzymatic cleavage, were compared to those obtained using conditions more favorable (19) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C.; Manz, A. Science 1993, 261, 895-897. (20) Wooley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (21) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113. (22) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (23) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178. (24) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (25) Figeys, D.; Ning, Y.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (26) Xue, Q.; Dunayevskiy, Y. M.; Foret, F.; Karger, B. L. Rapid Commun. Mass Spectrom. 1997, 11, 1253-1256. (27) Xu, N.; Lin, Y.; Hofstadler, S. A.; Matson, D.; Call, C. J.; Smith, R. D. Anal. Chem. 1998, 70, 3553-3556. (28) Figeys, D.; Aebersold, R. Anal. Chem. 1998, 70, 3721-3727. (29) Figeys, D.; Gygi, S. P.; McKinnon, G.; Aebersold, R. Anal. Chem. 1998, 70, 3728-3734. (30) Figeys, D.; Lock, C.; Taylor, L.; Aebersold, R. Rapid Commun. Mass Spectrom. 1998, 12, 1435-1444. (31) Xiang, F.; Lin, Y.; Wen, J.; Matson, D. W.; Smith, R. D. Anal. Chem. 1999, 71, 1485-1490. (32) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (33) Bings, N. H.; Wang, C.; Skinner, C. D.; Colyer, C. L.; Thibault, P.; Harrison, D. J. Anal. Chem. 1999, 71, 3292-3296. (34) Li, J.; Thibault, P.; Bings, N. H.; Skinner, C. D.; Wang. C.; Colyer, C. L.; Harrison, D. J. Anal. Chem. 1999, 71, 3036-3045. (35) Lazar, I. M.; Sundberg, S. A.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 3627-3631. (36) Liu, H.; Felten, C.; Xue, Q.; Zhang, B.; Jedrzejewski, P.; Karger, B. A. Anal. Chem. 2000, 72, 3303-3310.
1734 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001
Figure 1. Schematic representation of the nano-ESI microchip. HVPS, high-voltage power supply.
for positive ion electrospray ionization at a relatively acidic pH. Proteins could be identified within 5-15 min of digestion from submicromolar concentration solutions. A major advantage of the approach is that it can be readily adapted to a multisample microchip device for high-throughput analysis. EXPERIMENTAL SECTION Microchips fabricated from glass were provided by Caliper Technologies Corp. (www.calipertech.com) (Mountain View, CA). A schematic diagram of the device utilized in this study is shown in Figure 1. Nanospray tips (20-50 µm i.d. × 360 µm o.d., 5 µm i.d. at the tip, ∼2 cm long, from New Objective, Cambridge, MA) were inserted into a 360-400-µm opening drilled into the top cover plate of the chip and were secured in place with a removable glue (E6000 adhesive, Eclectic Products). All channels on the chip were 20 µm deep and 60 µm wide at half-depth, and the length from the cross intersection to the point of insertion of the ESI tip was 2.8 cm. MS data were acquired with a Jaguar orthogonal extraction linear TOF instrument (Sensar Corp., Provo, UT). The curtain gas (nitrogen) was set to 500-700 mL min-1, and the interface was heated to 90 °C. The ESI voltage was supplied by the original TOFMS-ESI power source. Detailed operation of the microchip nanospray source has been reported.35 Briefly, the microchip reservoirs and channels are filled with buffer and the chip is positioned (vertical orientation of the chip with the nanospray tip placed at 2-3 mm away from the MS sampling orifice) in front of the entrance aperture of the TOFMS using a three-axis translational stage. Voltage (1300-1600 V) is applied to the chip through a platinum electrode inserted into one of the inlet reservoirs to generate the electrospray at the tip. Once a stable signal is obtained by adjusting the potential and position of the chip, the power supply is momentarily turned off and the buffer in the reservoirs exchanged with the analyte solution. Electrospray is then reestablished by applying the necessary potential to the platinum electrode. The Sensar microspray source was used to determine optimal digestion conditions. Samples were delivered to this source at 0.3 µL min-1 using a syringe pump (Harvard Apparatus, Inc., Holliston, MA). All mass spectra were acquired over a 0-6000 m/z range. The instrument was pulsed at 5000 Hz and up to 25 600 spectra were summed, resulting in a final acquisition/storage rate of ∼0.4-0.2 spectra s-1. Tryptic digests were prepared at room temperature (if not otherwise specified) in ammonium acetate solution or ammonium bicarbonate buffer, using (20 to 1)/1 (w/w) protein/trypsin ratios. The solutions were prepared by combining a 12.5 mM aqueous salt mixture (pH 6.5 for ammonium acetate, and pH 8.1 for ammonium bicarbonate) with methanol in a 4:1 ratio (v/v) to yield a final 10 mM concentration. These solutions were prepared fresh,
prior to use. Peptide mass fingerprinting for protein identification was accomplished by comparing the measured ions with the theoretically predicted ions using the PeptideMass software within the SWISSPROT database available on the Internet (http:// expasy.hcuge.ch/tools/peptide-mass.html). Blood samples (∼5 µL) were collected in 200 µL of isotonic NaCl solution, filtered through 0.22-µm Ultrafree-MC microcentrifuge cartridges (Millipore Corp., Bedford, MA), and rinsed with 200 µL of NaCl solution. Most of the red blood cells remained intact in the upper section of the microcentrifuge cartridge. Next, the erythrocytes were lysed with water or water/methanol (50: 50 v/v) and filtered though the same 0.22-µm microcentrifuge cartridge. The filtrate (containing the hemoglobin) was collected with a syringe, centrifuged through 10 000 or 30 000 NMWL Ultrafree-MC microcentrifuge cartridges, and rinsed twice with 200 µL of water/methanol (50:50 v/v). The hemoglobin was retained in the upper section of the centrifuge cartridge. The final solution was diluted to 4 mL in water/methanol (50:50 v/v), and a 100-µL aliquot of this solution was used for tryptic digestion in ammonium bicarbonate buffer. Hemoglobin S stabilized standards were centrifuged through 5000 NMWL microcentrifuge cartridges to remove the stabilizing agent. Peptide map control cytochrome c, bovine and human hemoglobin, and sequencing grade trypsin (unmodified from bovine pancreas) were purchased from Sigma (St. Louis, MO). Deionized water (18 MΩ‚cm) was obtained from a NANOpure water system (Barnstead Thermolyne Corp., Dubuque, IA). Ammonium acetate (99.999%), ammonium bicarbonate (99%), and glacial acetic acid were purchased from Aldrich (Milwaukee, WI), and methanol (HR-GC grade) was from EM Science (Gibbstown, NJ). RESULTS AND DISCUSSION Rapid analysis and identification of proteins may be expedited by a rapid method for enzymatic digestion. Conventionally, tryptic digests are performed in basic buffer (pH 8-8.5) at a slightly elevated temperature (37 °C) for up to 24 h using 1-5% enzyme to substrate (by weight). For subsequent ESI MS analysis, volatile buffers are preferred. To enable high-throughput processing, the digestion time should be substantially reduced, and accordingly, our goal in this investigation was to digest and analyze individual samples within a few minutes. A recent report has shown that proteins may be rapidly digested for subsequent MS analysis using a microcolumn packed with a solid support containing immobilized trypsin (i.e., Poroszyme resin).18 In this off-line approach, it was necessary to desalt the samples following digestion and prior to ESI. Losses of smaller peptides on the desalting column and of higher molecular weight peptides, attributed to adsorption on the digestion cartridge, were reported. Rapid digestion in solution using nanoelectrospray for reaction monitoring over an extended time course has also been reported.17 In this case, analyses were limited to model peptides at relatively high concentration. A relatively acidic solution (ammonium acetate at pH 6.5) commonly used to promote the formation and determination of positively charged tryptic fragments was examined and compared to data obtained in ammonium bicarbonate buffer at pH 8 where trypsin is most active. Several recent reports have shown that it is possible to electrospray and detect positively charged ions from high-pH solutions.37-43 The observation of MH+ ions from basic solutions has been coined “wrong-way-round” electrospray.42
These studies indicate that the relative intensity of protonated amino acids, peptides, or proteins may decrease by only 1 order of magnitude when the pH of the bulk solution is changed from acidic to basic. To our knowledge, a practical application of this phenomenon has not been previously demonstrated. Cytochrome c was used as a test substrate at varying proteinto-enzyme ratios (from 20:1 to 1:1). Products were generated at room temperature and monitored at specified time intervals after mixing the enzyme and protein or continuously by electrospraying the digestion solutions for up to 2 h following mixing. Some samples were also incubated for 24 h at 37 °C, prior to analysis. As expected the digestions proceeded most rapidly at the highest enzyme-to-protein ratio (1:1). For 20:1 protein/enzyme samples in ammonium acetate (data not shown), the intensity of the fragments increased substantially over the first 2 h of the digestion, but not all fragments visible at 24 h were detected at the end of the 2-h period. For a 10:1 substrate/enzyme mixture in this solution (data not shown), most all of the ions present in the 24-h digest were observed following 30 min of digestion. For the highest substrate to enzyme mixture (1:1), there was no major difference between the spectra acquired after 15 min of digestion or 24 h. The disadvantage of using high trypsin levels, however, is the presence of strong background ions. Panels A and B of Figure 2 respectively show the spectra obtained, using a conventional microspray source, for 1 and 10 µg mL-1 trypsin (corresponding to 10:1 and 1:1 substrate/enzyme ratio samples) in ammonium acetate after 24 h of incubation at 37 °C. While a relatively clean spectrum above 400 m/z was obtained for the lower concentration, the 10 µg mL-1 solution displayed a somewhat cluttered background. Ions at 4032+, 4532+, 5772+, 633+, 659+, 7583+, 805+, 10822+, and 11372+ (labeled “t” in the figures) were identified as trypsin autolysis products, while ions 186+, 196+, 262+, 338+, 344+, 588+, 888+, and 1071+ (denoted by “tc”) were other contaminants in the trypsin sample which were not identified. These ions may make it difficult to accurately assign and identify tryptic protein fragments should they overlap with those of interest. It should be noted, however, that, for proteins with a database entry, the identification of three to five fragments is often sufficient for positive identification.30 Panels A and B inFigure 3 respectively compare the spectra for a tryptic digest of 0.8 µM cytochrome c (prepared using a 1:1 mixture of protein to enzyme, 10 µg mL-1 trypsin), in ammonium acetate, 25 min after the reactants were mixed and introduced onto the chip, and in ammonium bicarbonate buffer, following 15 min of digestion. Flow rates through the microchip device were on the order of 20-30 nL min-1. Only ∼2 fmol of sample (cytochrome c) was consumed to produce each spectrum. The background was relatively high with the ammonium acetate (37) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207. (38) Kelly, M. A.; Vestling, M. M.; Fenselau, C. C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143-1147. (39) Wang, G.; Cole, R. B.; Org. Mass Spectrom. 1994, 29, 419. (40) LeBlanc, J. C. Y.; Wang, J.; Guevremont, R.; Siu, K. W. M. Org. Mass Spectrom. 1994, 29, 587-593. (41) Hiraoka, K.; Murata, J.; Kudaka, I. J. Mass Spectrom. Soc. Jpn. 1995, 43, 127. (42) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120-1130. (43) Konermann, L.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1998, 9, 12481254.
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Figure 2. ESI-TOF mass spectra of trypsin at 1 (A) and 10 µg mL-1 (B) in 10 mM ammonium acetate/methanol (8:2 v/v) following 24-h incubation at 37 °C. TOFMS acquisition: 5000 Hz, 25 600 summed spectra. Abbreviations: t, trypsin autolysis products; tc, trypsin contaminants; C, system contaminants; B, buffer-related ions.
solution, and the intensity of the tryptic fragment ions was low. The reaction was monitored continuously over 60 min, and the intensity of these ions was not found to increase significantly. In the ammonium bicarbonate buffer, the background was substantially lower and the intensity of the fragment ions was higher. In general, fragments with no missed cleavages, such as 5842+, 634+, 678+, 7352+, and 779+, increased preferentially over time, while fragments with one or two missed cleavages decreased in intensity. There were, however, no major differences between spectra collected at 15 min or 1 h of digestion, indicating that any decrease in signal resulting from electrospray from a basic solution is compensated by the kinetic advantage of performing the digestion under more favorable pH conditions. The sequence coverage for cytochrome c, which has 104 amino acids and yields 22 tryptic fragments with no missed cleavages, at 15 min was 90%. Sufficient signal for positive identification of the protein was also obtained after 7 min of digestion, using a 5:1 substrate-to-enzyme (1 µg mL-1 trypsin, 400 nM sample) mixture (Figure 3C). Only 100 amol of sample was consumed to generate this spectrum. A high acquisition rate (3.2 spectra s-1) was used to collect this latter spectrum to demonstrate the ability to rapidly acquire useful data, as would be necessary if a fast separation were performed on the microchip (to provide initial separation of the fragments, for example). Under these conditions, peak widths may be less than 1 s wide and injection volumes on the order of a few picoliters, necessitating the use of such rapid rates. All the proteins we analyzed on-chip using the ammonium bicarbonate buffer at elevated pH are listed in Table 1 along with a summary of the results obtained. About half of the digestion fragments for each of the proteins examined have calculated isoelectric points of less than 8 and thus were detected under “wrong-way-round conditions”. The tryptic fragments for glucagon 1736 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001
Figure 3. Microchip-nano-ESI-TOF mass spectra of tryptic digests of cytochrome c. Conditions: cytochrome c (0.8 µM)/trypsin (1:1) in (A) 10 mM ammonium acetate containing 20% methanol, 25-min digestion and (B) in 10 mM ammonium bicarbonate containing 20% methanol, 15-min digestion. TOFMS acquisition: 5000 Hz, 25 600 summed spectra. Spectrum in (C) for 0.4 µM cytochrome c/trypsin (5:1) in 10 mM ammonium bicarbonate containing 20% methanol, 7-min digestion. TOFMS acquisition: 5000 Hz, 1600 summed spectra, 3.2 spectra s -1. Abbreviations are the same as in Figure 2; 8032+ ion from (T17-T20) or (T18-T21), denoted by *.
were detected in a couple of minutes, essentially immediately following sample loading, electrospray establishment, and the time for the sample to migrate to the tip. The spectrum for bovine hemoglobin is shown in Figure 4 and was produced from less than 10 fmol of sample consumed, with a total amount of 20 pmol introduced into the 5-µL microchip reservoir. The sequence coverage was 97% following 10 min of digestion (Table 2). Only 2 small fragments were not observed out of a possible 32 fragments (286 total amino acids) with no missed cleavages: 288+ (T10) from the R and 246+ (T3, T7) from the β-chain. Fragments with one missed cleavage that include some of these fragments were detected, however (i.e., 5442+, R T10 + T11; 5892+, β T2 + T3; and 6642+, β T3 + T4). It should be noted as well that in this case we dealt with a mixture of two proteins (i.e., R- and β-hemoglobin). We also investigated the analysis of hemoglobin in human blood. As a major constituent (12-16 g/100 mL of whole blood or 33-36% weight/erythrocyte) it can be easily isolated from other
Table 1. Summary of Results Obtained Using the On-Chip Digestion ESI MS Protocol
a
protein
total AA
total no. of fragments
no. of identified fragments
digestion time, min
sequence coverage,a %
cytochrome c glucagon bovine R-hemoglobin bovine β-hemoglobin human R-hemoglobin human β-hemoglobin
104 29 141 145 141 146
22 4 14 18 14 15
17-21 4 12-14 16-17 9-12 10
7-15 2 10 10 11-15 11-15
88-95 100 95-100 95-98 74-80 70
Includes fragments with one missed cleavage.
Table 2. Observed Fragments in the Bovine Hemoglobin Tryptic Digest (r- and β-Chains) and Comparison with the Data Base Output (SWISSPROT)a m/z measd (MH+, MH22+, MH33+)
tryptic fragment
no. of missed cleavages
peptide sequence
990.5053+ 879.4943+ 789.7693+ 917.4072+ 765.3612+ 640.3642+ 562.2842+ 551.3452+ 544.3052+ 1071.484+ 818.435+ 703.431+ 673.413+ 532.291+ 469.253+ 417.208+ 338.153+
2969.609 2636.379 2367.194 1833.892 1529.734 1279.726 1123.658 1101.626 1087.626 1071.554 818.441 703.398 673.424 532.288 469.252 417.246 338.182 288.203
R-Hemoglobin 100-127 (T12) 69-92 (T9+T10) 69-90 (T9) 41-56 (T6) 17-31 (T4) 128-139 (T13) 57-68 (T7+T8) 1-11 (T1+T2) 91-99 (T10+T11) 32-40 (T5) 93-99 (T11) 1-7 (T1) 62-68 (T8) 12-16 (T3) 57-61 (T7) 8-11 (T2) 140-141 (T14) 91-92 (T10)
0 1 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0
LLSHSLLVTLASHLPSDFTPAVHASLDK AVEHLDDLPGALSELSDLHAHKLR AVEHLDDLPGALSELSDLHAHK TYFPHFDLSHGSAQVK VGGHAAEYGAEALER FLANVSTVLTSK GHGAKVAAALTK VLSAADKGNVK LRVDPVNFK MFLSFPTTK VDPVNFK VLSAADK VAAALTK AAWGK GHGAK GNVK YR LR
824.3713+ 1045.4932+ 876.9362+ 739.4572+ 711.8922+
2471.202 2089.953 1752.899 1477.802 1422.726 1391.662 1328.717 1274.726 1265.830 1225.625 1177.680 1177.673 1101.553 1098.558 1097.53 950.509 821.407 740.394 465.246 412.230 319.140 246.181 246.181 147.113
β-Hemoglobin 82-103 (T12+T13) 40-58 (T6) 1-16 (T1+T2) 132-145 (T17+T18) 120-131 (T16) 82-94 (T12) 17-29 (T3+T4) 30-39 (T5) 104-115 (T14) 65-75 (T9+T10) 132-143 (T17) 8-18 (T2+T3) 19-29 (T4) 95-103 (T13) 66-75 (T10) 8-16 (T2) 1-7 (T1) 76-81 (T11) 116-119 (T15) 61-64 (T8) 144-145 (T18) 17-18 (T3) 59-60 (T7) 65-65 (T9)
1 0 1 1 0 0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0
GTFAALSELHCDKLHVDPENFK FFESFGDLSTADAVMNNPK MLTAEEKAAVTAFWGK VVAGVANALAHRYH EFTPVLQADFQK GTFAALSELHCDK VKVDEVGGEALGR LLVVYPWTQR LLGNVLVVVLAR KVLDSFSNGMK VVAGVANALAHR AAVTAFWGKVK VDEVGGEALGR LHVDPENFK VLDSFSNGMK AAVTAFWGK MLTAEEK HLDDLK NFGK AHGK YH VK VK K
664.8662+ 637.8702+ 633.3952+ 613.2652+ 589.3222+ 589.3222+ 551.3452+ 549.7262+ 549.2632+ 950.487+ 821.401+ 740.396+ 465.255+ 412.236+ 319.157+ 147.127+ aAll
m/z calcd (MH+) database entry
fragments with no missed cleavages are listed.
components that are present in low concentration (i.e., proteins, lipids, fatty acids, carbohydrates, amino acids, nucleotides, vitamins, and electrolytes).45,46 Normal human adult hemoglobin is a tetramer formed by two R-globin and two β-globin chains. More than 600 variants have been identified, to date, of which 95%
represent single amino acid substitutions on one of the chains.47 In the case of sickle cell anemia, for example, the glutamic acid in position 6 in the β-chain is replaced by valine. Given the serious consequences of these mutations on human health, early detection of abnormalities is essential and the development of new instruAnalytical Chemistry, Vol. 73, No. 8, April 15, 2001
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Figure 4. Microchip-nano-ESI-TOF mass spectrum of a tryptic digest of bovine hemoglobin. Conditions: substrate (4 µM)/trypsin (5:1) in 10 mM ammonium bicarbonate containing 20% methanol, 10-min digestion. TOFMS acquisition: 5000 Hz, 25 600 summed spectra.
Figure 5. Microchip-nano-ESI-TOF mass spectrum of a tryptic digest of 0.24 µM human hemoglobin extracted from blood. Conditions: as in Figure 4 following 15-min digestion. Abbreviations: t, trypsin autolysis products; tc, trypsin contaminants.
mentation and techniques that would enable high-throughput analysis for screening of newborns for known/unknown variants are desirable. Separations and/or MS analyses of hemoglobin tryptic digests are frequently used to determine specific variants.48-52 The techniques that have been used, however, are either relatively slow, are lacking in specificity, or are not readily amenable to highthroughput analysis. A diluted blood sample, filtered to remove particulates and low molecular weight substances, was digested on-chip and analyzed (Figure 5). The concentration of hemoglobin in the extracted blood sample was ∼0.6 µM (R- + β-chains), and a 5-µL aliquot was used for analysis (∼3 pmol of hemoglobin introduced onto the microchip). Sequence coverage of 70% was attained after 15 min of digestion (see Figure 6 for the tryptic map). The reduced coverage in comparison with that obtained for bovine hemoglobin (44) Lazar, I. M.; Jacobson, S. C.; Foote, R. S.; Ramsey, J. M.; Ramsey, R. S. Proceedings of the 47th ASMS Mass Spectrometry and Allied Topics, Dallas, TX, June 13-17, 1999. (45) Surgenor, D. MacN., Ed. The Red Blood Cell; Academic Press: New York, 1974. (46) Stevens, M. L. Fundamentals of Clinical Hematology; W. B. Saunders Co.: Orlando, FL, 1997. (47) Shackleton, H. L.; Witkowska, H. E. Anal. Chem. 1996, 68, 29A-33A. (48) Witkowska, H. E.; Green, B. N.; Morris, M.; Shackleton, C. H. L. J. Mass Spectrom. Rapid Commun. Mass Spectrom. 1995, S111-S115. (49) Hofstadler, S. A.; Swanek, F. D.; Gale, D. C.; Ewing, A. G.; Smith, R. D. Anal. Chem. 1995, 67, 1477-1480. (50) Hofstadler, S. A.; Severs, J. C.; Smith, R. D.; Swanek, F. D.; Ewing, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 919-922. (51) Li, M. X.; Wu, J.-T.; Liu, L.; Lubman, D. M. Rapid Commun. Mass Spectrom 1997, 11, 99-108. (52) Houston, C. T.; Reilly, J. P. Anal. Chem. 1999, 71, 3397-3404.
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is likely due to the hydrophobic core of human hemoglobin. Previously reported insoluble fragments are RT12, RT13, βT10, βT11, and βT12.51,52 The RT10 fragment was detected in our spectrum possibly as a result of the methanol in the digestion mixture. We did not attempt any further treatment of the digest to detect the other fragments. Some additional low-mass fragments were not observed and may not have been transmitted by the rf-only quadrupole of the instrument. The concentration of hemoglobin used in this analysis was lower than typically used in our optimization tests, although sample limitation is generally not a problem for this particular protein. The sample concentration can be readily adjusted according to need, since hemoglobin is abundantly available. A sickle cell hemoglobin (hemoglobin S) standard was also analyzed and could be easily distinguished (Figure 7). The 4762+ ion (βT1) in the normal hemoglobin digest is absent in the sickle cell digest. Unfortunately, the 4612+ ion (βT1) in the sickle cell digest could not be detected because of an interfering contaminant peak from a stabilizing agent in the standard. This contaminant was not completely removed by filtration through a low molecular weight cutoff filter. Although additional studies are required to determine the general applicability of this rapid microchip digestion/ESI TOFMS method for detecting hemoglobin variants, the results indicate that the technique is suitable for high-throughput processing. Only a minimal amount of pretreatment was required to cleanup whole blood prior to analysis. Microchip devices with multiple sample reservoirs may be automatically loaded and the samples digested in parallel. They may then be electrosprayed sequentially by applying the voltage consecutively from one inlet to the next, in a fashion similar to that previously reported.24,36 If spectra are collected under these conditions and allowing ∼15 s of MS analysis time per sample (switching to a new channel, establishing the electrospray, and collecting the spectrum), the total analysis time for 96 samples in a multiple channel microchip would be ∼25 min. Analysis time could also be somewhat reduced using immobilized trypsin where higher enzyme/substrate ratios may be employed without increasing the background (from trypsin autolysis products, for example).18 On-chip protein digestion on a packed trypsin bed has been recently demonstrated,53 although, significant improvements in digestion time were obvious only for small polypeptides. Moreover, a syringe pump, connected to the microchip, was necessary to transport the protein solutions through the bed and multiple, manual buffer exchanges were required. The major advantage of our approach lies in its simplicity. Consistent results are produced quickly from lowconcentration samples, and fluids are manipulated solely by the application of voltages. The future focus of our work will be directed toward developing a high-throughput microchip platform and on-line sample processing strategies to further reduce analysis times. CONCLUSION We have examined a method for rapid proteolytic digestions on a microchip with on-line TOFMS detection of the proteolytic fragments. The digestion is performed directly on the chip in an electrosprayable buffer at basic pH and at substrate/enzyme ratios (53) Wang, C.; Oleschuk, R.; Ouchen, F.; Li, J.; Thibault, P.; Harrison, D. J. Rapid Commun. Mass Spectrom. 2000, 14, 1377-1383.
Figure 6. Tryptic map of human hemoglobin.
that promote rapid cleavage. Positively charged fragments were
Figure 7. Mass spectra (expanded views) of digests of (A) normal human hemoglobin and (B) hemoglobin S. Conditions as in Figure 4. Abbreviatons: C, stabilizer contaminant from the sickle cell standard.
detected from low-concentration samples using wrong-way-round ESI conditions. Sample is delivered continuously through the
microchip and the attached nanospray tip at low flow rates of 2030 nL min-1 without any external assistance (pumping or pressurization devices). Additional sample manipulations such as desalting or chromatographic separation prior to ESI MS, as often performed in conventional procedures, were not required for analysis. A sufficient number of tryptic fragments to match with database entries and allow identification of proteins may be generated in less than 10 min from small quantities of starting material (i.e., low-femtomole or subfemtomole per spectrum, or low-picomole or subpicomole per analysis). Total sample requirements may be easily reduced by decreasing the volume of the sample reservoirs on the microchips. The procedure is amenable to automated high-throughput analysis using a parallel channel microchip platform with multiple sample ports.
ACKNOWLEDGMENT This research was sponsored by the National Cancer Institute under Grant IR33CA83238. Oak Ridge National Laboratory is managed by UTsBattelle, LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR22725. I.M.L. was supported through an appointment to the ORNL Postdoctoral Research Associates Program, administered by ORISE and ORNL. We thank Caliper Technologies Corp. for supplying the microchips.
Received for review December 4, 2000. Accepted February 6, 2001. AC001420+
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