Anal. Chem. 1995, 67,1705-1710
Peptide Mapping by CNBr Degradation on a Nitrocellulose Membrane with Analysis by Matrix-Assisted Laser Desorption/lonization Mass Spectrometry Jian Bai, Mark 0. Qian, Yanhui Liu, Xiaoli Liang, and David M. Lubman* Department of Chemistty, The University of Michigan, Ann Arbor, Michigan 48109
Nitrocellulose is examined as a membrane substrate for peptide mapping by CNBr digestion using MALDI MS analysis. Using this methodology, proteins electroblotted from SDS-PAGE can be p d e d on the membrane by washing and further characterized by in situ chemical digestion followed by MALDI MS analysis. It is demonstrated that nitrocellulose is versatile in such analyses due to its excellent retention of both small and large fragments and the wide range of fkagments that appear in MALDI MS. This is particularly advantageous in cases where multiple reactions and washing steps are involved. The analysis is found to be improved by ikst eluting the digest from the membrane, since interaction of the matrix with nitrocellulose may degrade the MALDI MS performance. One- and two-dimensional polyacrylamide gel electrophoresis (PAGE) remain widely used techniques for the separation and molecular weight determination of proteins because of their simplicity and general applicability.1 The subsequent transfer of proteins separated from SDS-PAGE onto solid supports, such as membranes, can then be used for further microisolation, purification, and characterization of proteins. In particular, the specificity with which membranes bind proteins allows the effective removal of contaminants or buffers required in protein sample handling and electrophoresis,resulting in proteins suitable for further characterization, such as peptide mapping and sequencing. The general approach for partial or complete structural analysis of proteins involves digestion of the purified proteins with selected endoproteinases, chemical cleavage methods, or both, followed by fractionation of the fragments generated. The resulting fragments can then be used to obtain peptide maps for protein identification or the sequence of the p r ~ t e i n . ~ - ~ Although PAGE can be used for molecular weight determination, the accuracy of the measurement may often be on the order of several percent. Thus, identification based upon mass spectrometry has been incorporated into the above strategy for peptide mapping and sequence verification. The recent advent of novel (1) Sfrahler,J. R; Kuik, R;Hanash, S. M. In Protein Structure: A Pructicul
Approch Creighton,T. E., Ed.; IRL Press: New York, 1989; pp 65-93. (2) Aebersold, R In Advances in Electrophoresis;Chrambach, A,, Dunn, M. J., Radola, B. J., Eds.; VCH-Verlag: Weinheim, Germany, 1991;Vol. 1,pp 81168. (3) Bauw, G. W.; Van Damme, S.; h y p e , M.; Vekerchove, B. G.; Raltz, G. P.; Lauridsen, J. B.; Celis, J. E.Proc. Nutl. Acad. Sci. U.S.A. 1989,86,77017705. (4) Abersold, R H.; Leavitt, J.; Saavedra, R A; Hood, L. E.; Kent, S. B. H. Pmc. Natl. Acad. Sci. U.S.A. 1987,84,6970. 0003-2700/95/0367-1705$9.00/0 0 1995 American Chemical Society
ionization techniques, electrospray ionization @SO5and matrixassisted laser desorption/ionization (MALDI),6,7 has made such analysis by mass spectrometry possible for proteins and large peptide fragments that result from chemical digestion. In particular, MALDI MS has quickly become an important tool for analyzing large biomolecules, because of its sensitivity, mass range, and capability of analyzing complex mixtures. The tolerance of this technique toward certain contaminants also makes it a first choice for initial screening and characterization of proteins before more extensive purification and manipulation. In recent work, it has been shown that proteins bound to membranes are amenable to chemical and enzymatic digestion followed by analysis using MALDI MS for the identification of proteins: peptide mapping:-l0 and localization of modifications.1° In work by ChaitlO and Henzel: protein digestion was performed on various membranes and the fragments were eluted from the membrane for MALDI MS analysis. They were able to demonstrate peptide mapping of proteins separated by two-dimensional electrophoresis*JO and were able to identify phosphorylation sites by use of this methodology.1° In other work, MALDI MS of the peptide fragments achieved directly from membranes and other active media has been investigated. In these studies, it was shown that proteins immobilized on membranes such as nitrocellulose," Nylon,lZand PVDF13-15could be analyzed directly by MALDI MS. In the present work, we examine the use of nitrocellulose as a membrane substrate for peptide mapping of CNBr digestion by MALDI MS. Nitrocellulose is commonly used as a membrane for electroblottingof gel-separated proteins and has been used in several studies for generating enzymatic or chemical cleavage peptide m a p ~ . ~InJ particular, ~ Aebersold and co-workers4used this strategy with nitrocellulose followed by analysis of the (5) Fenn. J. B.; Mann, M.; Meng, C. K; Wong, S. F.; Whitehouse, C. M. Science 1989,246, 64-71. (6) Karas, M.; Hillenkamp, F. Anal. Chem. 1988,60, 2299-2301. (7) Beavis, R C.; Chait, B. T. PYOC.Nutl. Acad. Sci. U.S.A. 1990,87,68736977. (8) Henzel, W. J.; Billeci, T. M.; Stults, J.T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U S A . 1993,90, 5011-5015. (9) Klarskov, K; Roepstorff, P. Biol. Mass Spectrom. 1993,22, 433-440. (10)Zhang, W.; Czemik, A J.; Yungwirth, T.;Aebersold, R; Chait, B. T. Protein Sci. 1994,3, 677-686. (11) Mock, K K; Sutton, C. W.; Cottrell, J. S. Rapid Commun. Mass Spectrom. 1992,6,233-238. (12) zaluzec, E. J.; Gage, D. A;Allison,J.; Watson, J. T.]. Am. SOC.Mass Spectmm. 1994,5,230-237. (13) Eckerson, C.; Strupat, IC; Karas, M.; Hillenkamp, F.; Lottspeich, F.; Electrophoresis 1992,13, 664-665. (14) Strupat, K; Karas, M.; Hillenkamp, F. Anal. Chem. 1994,66,464-470. (15) Vestling, M. M.; Fenselau, C. Anal. Chem. 1994,66, 471-477. (16) Montelaro, R C. Electrophoresis 1987,8, 432-438.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995 1705
fragments with reversed phase HPLC and SDS-PAGE. In their work, the peptide fragments were removed from the membrane for such analysis. In the work discussed herein, we examine nitrocellulose as a substrate for peptide mapping using a strategy similar to Aebersold4but with MALDI MS detection and compare nitrocelluloseto several other membranes including Immobilon-N and Immobilon-CD. We demonstrate that nitrocellulose is versatile in such analyses due to its excellent retention of both small and large fragments and the wide range of fragments that appear in the MALDI MS spectrum as compared to other membranes. The analysis is found to be improved significantlyby fist eluting the digest from the membrane, since interactions of the matrix with nitrocellulose may degrade the membrane and MALDI MS performance. EXPERIMENTAL SECTION Materials. Cytochrome c, myoglobin, ribonuclease A, and pcasein were obtained from S i a Chemical Co. (St. Louis, MO). PVDF-based membranes including Immobilon-N and ImmobilonCD were gifts from Dr. Janet Tice of Millipore Corp. (Bedford, MA). Nitrocellulose was obtained from Schleicher and Schuell (Keene, NH). 2,5Dihydroxybenzoic acid (DHB), caffeic acid (CA), sinapinic acid (SPA), and a-cyan&hydroxycinnamic acid (a-CHCA) were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used as matrices without further purification. Protein Blotting. Two methods of blotting were employed in this experiment. An initial effort to use dot blotting was tried in which proteins ranging from 0.5 to 1.0 pg in 50 mM NaHC03 were applied directly onto a piece of membrane of -4 x 4 mm2 in size. In the case of nitrocellulose and Immobilon-CD, no prewetting was performed before blotting because of the hydrophilic nature of these two membranes. Immobilon-N membrane was prewetted with methanol and subsequently washed with water before blotting according to the manufacturer’s protocol. The proteins adsorbed onto the membranes were allowed to dry and washed with water. For electroblotting of gel electrophoresis separated proteins, 0.5-1.Opg of proteins was applied on different lanes and separated using 1-D SDS-PAGE using the method of Laemmli.I7 The separated proteins were electroblotted onto nitrocellulose or WDF membranes using 50 mM sodium borate, pH 9.0, containing 5% (v/v) methanol (cathode) or 20% (v/v) methanol (anode) in a semidry blotting apparatus (SemiPhor, Hoefer) at a constant current of 1.3 mA/cm2 for 2 h. The protein bands on nitrocellulose membrane were visualized and marked by staining with Amino black (0.1% in 25% 2-propanol and 10% acetic acid) for 1 min and destaining for 30 min with several changes of the same solvent. In the case of the Immobilon-CD membrane, protein bands were visualized and marked by the Immobilon-CD staining kit, followed by destaining with pure methanol three times. The marked bands on the membrane were then carefully excised with a razor blade, cut into small pieces, and washed with water several times in a centrifuge vial before in situ CNBr digestion. Reduction and Alkylation. For proteins, particularly ribonuclease A, where multiple disulfide linkages remain intact after CNBr digestion, reduction of proteins adsorbed onto the membranes was performed by adding 5 p L of 0.1 M dithiothreitol @‘IT)and 25 p L of 0.4 M NH4HC03 at room temperature. After incubation for 30 min, 5 pL of 0.1 M iodoacetamide was added, and the reaction mixture incubated for another 30 min. Iodoacetamide was added after reduction of the disulfide bonds by D’IT to alkylate the resulting products, which are protein-CHzSH and 1706 Analytical Chemistty, VoL 67,No. 10, May 15, 1995
protein-CHzSCHzCONHz, to prevent the disulfide bonds from forming again. The reaction was stopped by withdrawing the reaction mixture, and the membrane was washed with water twice before CNBr digestion. Protein Digestion. The small pieces of membrane with the blotted protein were placed into a 0.6 mL polyethylene centrifuge vial with the addition of 10 pL of 0.5 M cyanogen bromide in 0.1 N HCl. The vial was flushed with dry nitrogen gas, sealed, and incubated in the dark for 1-2 h at room temperature. The reaction was stopped by withdrawing the liquid portion, and the membrane was either transferred to another vial for drying or the membrane was washed with several changes of deionized water before extraction of the CNBr fragments. The washing of the membrane after CNBr digestion enhances the sensitivity of the MALDI MS analysis considerably but at the expense of losing smaller fragments. Extraction of the membrane without washing improved the recovery of small fragments, but may result in a partially uninterpretable MALDI MS spectrum for the peptide fragments of proteins like /%casein. Extraction of the CNBr Fragments. The CNBr fragments were extracted from the membranes with 10 pL of (1) different solvent systems, such as watedacetonitrile (up to 2:3) and water/ 2-propanol (2:l) with the addition of TFA (up to 0.5%), and (2) MALDI matrix solutions1°in the above solvent systems, depending on the estimated amount of proteins on the membrane. a-CHCA, DHB, CA, and SPA were tested as matrices in this work. Sample Preparation for MALDI. The extract (2.5 pL) obtained with solvent extraction was applied onto the probe tip and allowed to evaporate to dryness, followed by addition of 2-3 pL of matrix before analysis. For extracts obtained with matrix solution, 2.5 pL of the extract was applied and dried on the probe tip before MALDI analysis. Mass Spectrometer. The TOF MS used in these studies was a m o d ~ e dWiley-McLaren design with the capability for highvoltage acceleration up to *20 kV (manufactured by R M. Jordan Co., Grass Valley, CA). A DCR 11 Nd:YAG laser system (Spectraphysics, Mt. View, CA) delivering 355 nm radiation was used for desorption. The laser beam was focused onto the probe tip at a 45O angle to the probe surface with a single 12.5 in. focal length quartz lens. The detector was a triple microchannel plate (MCP) detector with a Cu/Be conversion dynode, designed for postacceleration capability up to f 1 5 kV. The postacceleration stage enhances the efficiency for detection of heavy species but at the expense of the resolution. A resolution of -70 was available for fragments of ~ 3 0 0 0u, but the resolution was only -40 for larger fragments. Data were recorded using a Lecroy 9400 digital oscilloscope and subsequently transferred to an IBM PC/AT for processing. The intact molecular ion (MH+) and the corresponding doubly charged ion (MHz2+)were used for initial mass calibration in cases where these ions were present in the spectrum. The mass measured for smaller fragments using this approach is systematically lower than the value expected. After a mass spectrum was acquired, an internal standard of lower mass, such as gramicidin S, substance P, or bovine insulin, was added to the probe tip immediately for another analysis to establish the mass scale. The calibrated masses were then used in the original spectrum to redefine the mass scale. For spectra that have no intact molecular ions, two internal standards were added and the mass was calibrated in the same manner.
RESULTS AND DISCUSSION The mass spectra in this work were acquired using irradiation by the third harmonic (355 nm) of a Nd:YAG laser (Model DCR11,Spectraphysics) and each spectrum consists of an average of 200 single-shot spectra. Each spectrum consists of less than half of the amount of sample originally used in the gel separation, where only part of the material electroblotted onto the membrane is digested. Then approximately 20% of the final digest extract was applied onto the probe tip for MALDI MS analysis. The matrix used in this work is a-CHCA in 50%acetonitrile and 50% deionized water containing 2%'ITA All the spectra shown were obtained with proteins electroblotted to membranes after 1-D SDS gel separation, except Figure 4b, which was digested in solution. Membrane Evaluation. Membranes based on PVDF, nitrocellulose, nylon, and glass fibers are currently used for protein immobilization.16 Some of the important considerations in standard protein immobilization procedures include the efficiency of protein transfer from gel to the membrane and the recovery of proteins afterward, which are mainly affected by the binding capacity of the membrane used. For most of these membranes, the binding capacity is sufficient to accommodate the amount of samples normally found in SDS-electrophoresis. The recovery efficiency of the immobilized proteins though is often low with eluting solvents. Chemical/proteolytic digestion methods are routinely employed to generate fragments directly on the membrane, which result in greatly improved recovery of the peptide fragments. The fragments generated are then used for peptide mapping or sequencing, often after HPLC fra~tionation.~ Mass spectrometry, particularly FAB MS, has been used for the analysis of smaller Larger fragments have not been accessible to mass spectrometric analysis until recently with the introduction of ESI and W D I . The capability of MALDI MS for mixture analysis can provide a fast and sensitive method for this task. Another important factor that needs to be considered is the retention of proteins during sample handling, such as digestion and washing, which are essential for obtaining pure samples without HPLC purification and without significant loss of sample. This is particularly true for MALDI MS analysis of low-level samples. The membranes studied in this work, Immobilon-CD and Immobilon-N, are PVDF membranes having a relatively higher cited binding capacity for proteins as compared to the nitrocellulose membrane . However, MALDI MS analysis of the CNBr fragments obtained from proteins immobilized on these membranes revealed that there was a severe loss of CNBr fragments with a mass lower than 1500 u following digestion and washing of the membrane in the case of Immobilon-CD and Immobilon-N. However, fragments as low as 700 u were still retained on nitrocellulose following the same procedure and detected by MALDI MS and were not affected by additional reaction steps such as reduction/alkylation and additional washing. Figure 1shows the MALDI MS spectra of CNBr fragments of ribonuclease A (corresponding to -18 pmol applied on gel) immobilized on Immobilon-CD and on nitrocellulose. Fragment RA1, the most intense peak corresponding to residue 1-13 with a molecular weight of 1499 after digestion on nitrocellulose (17)Laemmli, U. K Nature 1970,227,680-683. (18)Camillen, P.;Haskins, N. J.; New, A. P. Rapid. Commun. Mass Spectrom. 1989,3,440-442. (19)Hall, S. C.;Smith, D.M.; Masiarz, F.R; Soo, V. M.; Tran, H.M.; Epstein, L. B.; Burlingame,A. L.Proc. Natl. Acad. Sci. U.S.A. 1993,90,1927-1931. (20) Mahrenholz, A M.; Carafoli, E.; Gonnet, G. J Biol. Chem. 1993, 268, 13015-13018.
100
l
80
-
eo
-
~
l
~
l
'
l
-
(RAZ+RA3+RA4+RA5)'
0.0
5 . 0 ~ 1 0 ~ l.0x104
1 . 5 ~ 1 0 ~ 2 . 0 ~ 1 0 ~ 2.5~10'
3.0~10'
mh Figure 1. MALDI mass spectra of CNBr fragments of ribonuclease A digested on (a) nitrocellulose membrane and (b) Immobilon-CD membrane taken using an a-CHCA matrix and laser radiation at 355 nm.
membrane, was totally absent in the spectrum obtained from digestion on either Immobilon-CD or Immobilon-N membranes. Figure 2 shows the MALDI mass spectra of the CNBr fragments of p-casein (corresponding to -10 pmol applied on gel) digested on Immobilon-CD and nitrocellulose. In the spectrum, small fragments below 1500 u are absent when Immobilon-CD is used, and much larger fragments, such as fragments CA6 (residues 167-185) with a mass of 3191 u and CA5 CA6 (residues 145185) with a mass of 4555 u, are also missing. However, other peaks appearing in the spectra have similar or improved intensity when Immobilon-CD is used, such as peaks CA7, CA4 CA5 and CA1. There was a similar loss of fragments also observed when Immobilon-N was used. The loss of fragments of low and high mass from ImmobilonCD and Immobilon-N may be due to the fact that both membranes are positively charged, while nitrocellulose may be negatively charged at the pH of these experiments. The acidic conditions during CNBr reaction rupture the ionic interactions in the case of using Imm~bilon-CD~~~~ and Immobilon-N membranes, so that some fragments, particularly the smaller fragments, were selectively released, while the much larger fragments (e.g., CA6 and CA5 + CA6) could be either selectively released or strongly retained. A similar observation has also been made by Chait et al."J using an Immobilon-CD membrane for CNBr digestion of synapsin I. Further experiments conducted in our laboratory involving the recovery of fragments in the liquid reaction portion and the water used for washing, and a comparison of the fragments extracted from unwashed and washed membranes, confirmed that those smaller fragments were lost during both
+
+
(21)Patterson, S.D.;Hess, D.;Yungwirth, T.;Aebersold, R Anal. Biockem. 1992, 202,193-203.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1707
~
s7 loo
loo
-.-p *
0.0
5.0~10'
1.ox1' 0
1.5~10'
-:
0.00
250x103
500x103
7.50~103
i.ooxi 04
2.50~10~
5 . 0 0 ~0 '1
7.50~10~
1.00~10~
100
80
60
40
20
0 5.0~10'
0.0
1 .ox1o4
1.5~10'
0.00
mlz Figure 2. MALDI mass spectra of CNBr fragments of ,&casein digested on (a) nitrocellulose membrane and (b) lmmobilon-CD membrane taken using an a-CHCA matrix and laser radiation at 355 nm.
mlz Figure 3. MALDI mass spectrum of CNBr fragments of ,&casein digested on (a) nitrocellulose and (b) Immobilon-CD membrane taken using an a-CHCA matrix and laser radiation at 355 nm. The CNBr fragments were extracted from the membrane without washing.
digestion and the washing step. Larger fragments, including CA6 and CA5 CA6, were not detected in either the digestion solution or the washing water. This suggests that the absence of larger fragments from the Immobilon-CD membrane is due to the poor extraction efficiency of these fragments. There is no significant difference between dot-blotted and electroblotted proteins using these membranes, even though larger fragments appeared to have slightly lower efficiency from MALDI MS analysis in the case of electroblotted proteins. A serious problem observed in this experiment is that even though direct extraction of the CNBr fragments from membranes without washing improved the recovery of smaller fragments, spurious and uninterpretable peaks arise in the MALDI MS spectrum for p-casein. Panels a and b of Figure 3 show the MALDI mass spectra of the CNBr fragments of k a s e i n (corresponding to -10 pmol applied on gel) on nitrocellulose and Immobilon-CD without washing after the digestion. In the case of the Immobilon-CD, smaller fragments previously absent with washing are present, but most of the larger CNBr fragments are replaced by peaks that seem to be uncharacteristic of that expected from CNBr digestion. No such changes were observed for other proteins used in this work with the same procedure, even though the additional washing step immediately after digestion considerably enhanced the MALDI MS analysis, especially for low level samples (see below). The origin of these unexpected peaks is not clear, but they consistently appear if the washing step is omitted. This may be caused by the CNBr residues on the membrane that can interact with the peptides during subsequent sample handling or MALDI MS analysis. In comparison, in Figure 3a is the CNBr digest of Bcasein on nitrocellulose, where these uninterpretable peaks were not
observed and the spectrum obtained appears very similar to that of Figure 2a for the washed case. After washing, the intensity of the low-mass ions decreased; however, ions were still available for MALDI MS analysis in the low-mass range. A comparison of the CNBr digestion of horse heart myoglobin, ribonuclease A, and ,&casein in solution and on a nitrocellulose membrane was also performed by MALDI MS analysis. The digestion of pure proteins in solution appears to provide better sensitivity in MALDI MS analysis. However, since proteins are electroblotted onto membranes following SDS-PAGE and the proteins are often strongly bound to the membrane, the strategy of digestion in solution is often impractical. No significant difference was found with the amount of sample tested in the case of ribonuclease A and pcasein. Figure 4 shows the MALDI mass spectrum of CNBr fragments of myoglobin (correspondingto -7.5 pmol applied on gel) after digestion in solution and on a nitrocellulosemembrane. The MALDI MS spectrum of the CNBr digest of myoglobin obtained in solution shows more fragmentation products than the membrane. This is expected due to the nonhomogenous extraction efficiency of different fragments from the nitrocellulose membrane. However, the MAID1 MS spectrum of myoglobin obtained after digestion on the nitrocellulose membrane in Figure 4 is very similar to that published earlier in ref 10, although the spectrum in ref 10 had a somewhat better S/N. Elution/Exiraction of CNBr-Generated Fragments for MALDI MS Analysis. Two procedures were adopted for the recovery of CNBr fragments from membranes. One of the most commonly used procedures involves the use of a 20-40% acetonitrile/water mixture. This procedure results in fragments that are virtually free of buffers or contaminants, suitable for analysis
+
1708 Analytical Chemistry, Vol. 67,No. 10, May 15, 1995
00
80
40
20
.-=
0.0
bl -
loo
5.0~10~
1.0~104
1.5~104
2.0~104
25x104
I : H 0.00
MGl'(1-55)
2.50~10'
5.00~10'
750x1' 0
1.OOXlO'
mlz
Flgure 5. MALDI mass spectrum of CNBr fragments of ribonuclease A alkylated and digested on nitrocellulose membrane taken using an a-CHCA matrix and laser radiation at 355 nm.
0.0
5.0~10~
l.0x104
1.5~10~
2.0~10'
2.5~10'
mlz
Figure 4. MALDI mass spectra of CNBr fragments of myoglobin digested on (a) nitrocellulose membrane and (b) in solution taken using an a-CHCA matrix and laser radiation at 355 nm.
by both MALDI MS and conventional methods. This method is the first choice since the fragments generated can also be examined by other methods without contamination from the matrix. Another procedure adopted1° involves the extraction of the fragments with matrix solution prepared in acetonitrile/water with the addition of TFA Of the matrices tested, a near-saturated solution of a-CHCA in 40-60% acetonitrile and water mixtures with 0.5-1.5% TFA worked well for all the membranes. The use of a CA matrix in the above solvent system causes severe degradation of the nitrocellulose membrane and is less effective for subsequent MALDI MS analysis. SPA and DHB are also less effective as matrices compared to a-CHCA a-CHCA in 30% 2-propanol/water with 1%TFA or a-CHCA in acetonitrile/Z propanoic acid/water (42:4) was also found to be effective for the proteins tested. There is no significant difference between the two extraction methods evaluated in terms of MALDI MS analysis, even though extraction directly with matrix solution is more convenient and appears more efficient if shorter incubation time is used. Although extraction of the CNBr fragments from nitrocellulose using acetonitrile/water mixture with acetonitrile as low as 20%is quite efficient for a 3-4 h incubating period at 37 "C, extraction with a-CHCA in the above solvent system under the same condition followed by MALDI MS analysis was not successful. This may reflect the complexity of the interaction between the peptide and the nitrocellulose. The addition of a-CHCA, a weak acid, may enhance the interaction of the peptide with the nitrocellulose, making the extraction less efficient, or it may simply be a matter of the concentration of the matrix in solution being too low. One of the problems involved with CNBr digestion, or other digestion methods in general, is that disulfide bonds are usually
retained, as in the case of ribonuclease A, where multiple disultide bonds exist. Direct CNBr digestion of ribonuclease A generated only two fragments (Figure l),as opposed to the expected five fragments. The use of a membrane allows the reduction/ alkylation to be performed on the membrane before CNBr digestion. Figure 5 shows the mass spectrum of the CNBr digest of ribonuclease A (corresponding to -9 pmol applied on gel) after reduction with DlT and alkylation with iodoacetamide. Cleavage is complete except for the M-M bond. One of the most attractive features of using a membrane in this case is that the washing step can be used after each chemical reaction, i.e., after reduction/ alkylation and then again after CNBr digestion. This effectively eliminates possible cross contamination between reactions and contamination at the final stage before MALDI MS analysis. Washing the membrane after CNBr digestion was found not to be essential in cases where a large amount of sample is digested, since a larger amount of matrix solution can be added to dilute the CNBr. However, the final washing step before extraction was found to be important if smaller amounts of samples are analyzed. Figure 6 shows the mass spectra of cytochrome c digest (corresponding to -4 pmol applied on gel) with and without the final washing step. As can be seen from Figure 6, washing considerably enhanced the quality of the mass spectrum. More importantly, washing also effectively avoids the occurrence of extraneous peaks in CNBr fragments for proteins like p-casein. Chemical/proteolytic digestion is widely used for generating fragments for internal sequencing, peptide mapping, protein identification, and modification detection. Protein identification based on molecular masses of two or more fragments from chemical/proteolytic digestion has been demonstrated.8 This requires mass determination to be as accurate as possible and the cleavage as specific as possible. The use of mass spectrometry, and MALDI MS in particular, can greatly facilitate this process. However, the general utility of this approach for chemical digestion, such as CNBr digestion, may be dif6cult to implement, either because of the poor documentation of the possible modifications of proteins or because of modifications that may occur during chemical digestion. Table 1 lists the predicted and measured molecular masses of CNBr fragments of Bcasein, ribonuclease A, and alkylated ribonuclease A, which are relevant for the following discussion. Analytical Chemistfy, Vol. 67,No. 10, May 15, 1995
1709
Table 1. Calculated and Measured Molecular Mass of CNBr Fragments of Some Proteins
CNBr framents
0.0
100
5.0~10’
i.ox1o4
1 . 5 ~O4 1
I
I
I
2.0~10~ 1
-
0.0
i.ox1o4
5.0~10’
1.5x104
2.0~10~
mlz Figure 0. MALDI mass spectra of CNBr fragments of cytochrome c digested on nitrocellulose membrane (a) without washing and (b)
with washing taken using an a-CHCA matrix and laser radiation at 355 nm.
As shown in Table 1, the mass accuracy varies dramatically from f0.07 to 1.87%, but is well divided into two general categories, the ones with absolute error within A10 u and the ones above f29. It appears that the large differences between the measured and predicted masses in some cases such as fragments CA1 and RA2 RA3 RA4 RA5 may be caused by adduct formation. For the RA2 RA3 RA4 RA5 fragment, the measured mass of 12 168 u is closer to the value of 12 155 u, assuming the three methionine residues remained intact during digestion, or for CA1, the difference maybe due to phosphorylation.22 The fragments CA5 CA6, ARAl ARA2, and ARAl ARA2 ARA3 may be present with one uncleaved methionine converted to homoserine, which should have a predicted mass difference of -30 u. Fragments CA7 and ARA4 may represent other variations, e.g., ARA4 may be present with only three cysteine groups alkylated, instead of the predicted four. Such discrepancies have also been observed in other w0rk,9,~~ and are not well resolved at present. There is a need here to reevaluate the various chemical digestion methods using mass spectrometric measurements and to develop databases and improved strategies for searching these databases for more accurate and faster identification of proteins and for structural veacation. Nevertheless, incorporating MALDI MS with chemical dgestion methods through the use of the nitrocellulose membrane provides a fast and convenient method for direct analysis of such digests, and
+
+
+ +
+
+
+
+
+
+
Bcasein CA1 (1-93) cA2 (94-102) CA3 (103-109) CA4 (110-144) CA5 (145-156) CA6 (157-185) CA7 (186-209) CA5 CA6 CA4 CA5 CA6 CA7 ribonuclease A RA1 (1-13) RA2 (14-29) RA3 (30-30) RA4 (31-79) RA5 (80-124) RA2 RA3 RA4 RA5 alkylated ribonuclease A ARAl (1-13) ARA2 (14-29) ARA3 (30-30) ARA4 (31-79) ARA5 (80-124) ARAl+ARA2 ARAl+ARA2+ARA3
predicted measd massu mass error error (%)
+ + +
10504 900 792 4020 1334 3191 2616 4555 5394 5837
10700 896 790 4017 1330 3185 2658 4526 5385 5841
196 -4 -2 -3 -4 -6 42 -29 -9 4
1.87 -0.44 -0.25 -0.07 -0.30 -0.19 1.61 -0.64 -0.17 0.07
1497 ndb nd nd nd 12168
-2
-0.13
+
1499 1605 101 5450 4869 12011 1499 1662 101 5682 5043 3192 3323
1500 1665 nd 5627 5048 3166 3289
+
+
1710 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1.31
1
0.07 0.18
3
-55
-0.97
5 -26 -34
-0.81 -1.02
0.10
The mass was calculated by assuming the Gterminal of methionine was converted to homoserine lactone after CNBr cleavage. nd, not detected. (I
for most of the fragments the measured mass matches reasonably well with the expected values. CONCLUSION MALDI MS can provide a general means for the characterization of proteins electroblotted from PAGE onto membranes. Immobilized proteins can be purified on these membranes simply by washing and further characterized by incorporating chemical digestion methods directly on the membrane. A nitrocellulose membrane with moderate binding capacity and excellent retention for both small and large fragments is advantageousin cases where multiple reaction and washing steps are used. It effectively eliminates possible cross contamination between reactions and contamination at the final stage before MALDI MS analysis. The incorporation of MALDI MS with the membrane immobilization technique has provided a fast and effective means of peptide mapping based on chemical digestion without the use of conventional separation or fractionation methods. ACKNOWLEWMENT
This work received support from the National Institutes of Health under Grant lROlGM49500 and partial support from the National Center for Human Genome Research under Grant 1R21HG006850W. Received for review December
(22) Lao, P.C.;Leykam,J.;Andrews, P. C.; Gage, D. A; Allison, J.Anal.Bwchem. 1994,219, 9-20. (23) Andrews, P. C.; Men, M. H.; Vestal, M. L.; Nelson, R W. In Techniques in Protein Chemistry;Angeletti, R H., Eds.; Academic Press, Inc.: New York, 1992; pp 515-523.
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5, 1994. Accepted March
7,1995.@ AC9411755 Abstract published in Advance ACS Abstracts, April 15, 1995.
