Nature Biotechnology
22, 1291 - 1296 (2004)
Published online: 7 September 2004; | doi:10.1038/nbt1011
Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysisHongying Zhong, Ying Zhang, Zhihui Wen
& Liang LiDepartment of Chemistry, University of Alberta, Chemistry Center W3-39, Edmonton, Alberta T6G 2G2, Canada.
Correspondence should be addressed to Liang Li liang.li@ualberta.caThe characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.
Protein modifications, such as post-translational modifications, can be characterized only by examining a protein directly. At present, reading the amino acid sequence of a protein is performed by the Edman degradation method and increasingly by tandem mass spectrometry (MS/MS)1,
2,
3,
4. Other MS-based techniques, such as ladder sequencing5,
6,
7,
8,
9,
10,
11,
12, in-source fragmentation13,
14 and chemical derivatization15,
16, have been reported for sequencing peptides with varying degrees of success. Compared to the Edman method, the MS approach has the advantages of having high sensitivity and generating structural information on modifications. However, to map the sequence of an entire protein for examining all possible modifications, one often needs to produce, detect and sequence many short, partially overlapping peptides, which can be very difficult.
Our protein sequencing technique is based on the mass analysis of polypeptide ladders (MAP) of a protein after it has been subjected to brief hydrolysis in HCl with the assistance of microwave irradiation (Fig. 1). We note that previous attempts using enzymes3, modified Edman chemistry6 and acids7,
8,
9,
10,
11,
12 to produce peptide ladders were not generally applicable to sequencing peptides because of a lack of specificity (that is, the methods generated internal peptides along with some peptide ladders) and universality (that is, most peptides failed to generate any ordered peptide ladders for sequence reading). In contrast, the MAP sequencing technique allows direct sequencing of peptides and even proteins with high sensitivity and speed (Fig. 1). Figure 2 shows the matrix-assisted laser desorption ionization (MALDI) spectra of 1 pmol cytochrome c hydrolyzed under different conditions (the expanded spectra in the m/z range from 2,500 to 6,000 are shown in Supplementary Fig. 1 online). Without microwave irradiation, a small number of polypeptides in the low-mass region were observed after the protein and HCl mixture was kept at room temperature (21 °C) for 5 min (Fig. 2a). These polypeptides were found to be exclusively N- and C-terminal peptides. If the mixture was kept for longer periods, e.g., 15 h, more intense terminal peptide peaks were observed (Fig. 2b). The speed of hydrolysis could be dramatically accelerated using microwave irradiation, possibly because of microwave-induced rapid heating and conformational or structural changes of the proteins along the peptide bonds. When the microwave radiation was applied for only a short duration (10 s), a number of peaks were detected at m/z below the molecular ion region and they were distributed across a broad mass range (Fig. 2c). As the irradiation time was increased, the intensities of these terminal peptide peaks also increased (Fig. 2d−f). Mass spectra displaying peaks over a broad mass range with good signal-to-noise ratios could be generated with microwave irradiation for 30 to 90 s. As the irradiation time was increased further, many internal peptides started to appear and the high-mass peptide peaks, along with the molecular ion peak, disappeared (Fig. 2g−i).
 | | |
Similar time dependencies to those shown in Figure 2 were observed for other proteins, such as human ubiquitin (MW, 8,565 Da) and horse apomyoglobin (MW, 16,952 Da) (data not shown). It appears therefore that when the protein hydrolysis process is properly controlled, e.g., using 6M HCl with microwave irradiation for 1 min, mass spectrometric analysis of the resulting hydrolysate generates a spectrum consisting of peaks exclusively from terminal peptides with no internal peptides, which makes the reading of protein sequence very easy. This somewhat surprising observation may be explained by considering the reaction rates of peptide bond breakage and the concentration differences among the intact protein, terminal peptides produced from the first hydrolysis process and internal peptides produced from the follow-up hydrolyses of the terminal peptides. With 10 s microwave irradiation, a number of terminal peptides have already been generated, but their signal intensities are much lower than that of the intact protein, indicating that the relative abundances of these terminal peptides to the intact protein are very low (Fig. 2c). As the irradiation increases from 10 to 60 s, more intense terminal peptide signals are generated, but their relative intensities to that of the intact protein are still low. Furthermore, it appears that all peptide bonds of the intact protein are cleaved once and only small variations in relative intensity of adjacent terminal peptides are found, which suggests that the reaction constant for peptide bond breakage is similar for all peptide bonds. Therefore, in the first hydrolysis process, an intact protein consisting of many peptide bonds is most likely to undergo parallel reactions by breaking any one of the peptide bonds once, to form, collectively, many terminal peptides. If only the first hydrolysis of the intact protein took place, the amount of an individual terminal peptide would equal the amount of the intact protein hydrolyzed divided by the number of peptide bonds broken in the protein and, at the early stage of hydrolysis where the intact protein concentration is high, the terminal peptide concentration would be expected to be very low. In reality, the terminal peptides formed early on in the hydrolysis process, e.g., those terminal peptides giving rise to the peaks shown in Figure 2c after 10 s microwave irradiation, could further hydrolyze to form shortened peptides including internal peptides (that is, follow-up hydrolysis). However, as indicated earlier and shown in Figure 2c,d, the amount or concentration of an individual terminal peptide generated early on in the hydrolysis process is relatively low compared to that of the intact protein. Because the hydrolysis reagents (that is, water and acid) are in excess, the acid hydrolysis rate of the intact protein or a terminal peptide is pseudo-first order in protein or peptide concentration, that is, v = k[C], where v is the reaction rate, k is the reaction constant, and [C] is protein or peptide concentration. Thus, at the early stage of hydrolysis, a terminal peptide is generated very quickly from the intact protein and only a small portion of it is further hydrolyzed. The net result of these reaction kinetics is that an excess amount of the terminal peptide is accumulated until its concentration is comparable to that of the intact protein. Beyond this time the terminal peptide will not be replenished from the hydrolysis of the intact protein as quickly and the concentrations of the internal peptides generated from the follow-up hydrolysis of the terminal peptide will build up. After 3 min microwave irradiation, terminal peptides generate similar peak intensities as that of cytochrome c and many internal peptides are observed (Fig. 2g). Once all intact protein molecules are consumed and shortly thereafter, the internal peptide peaks become the dominating feature of the spectra (Fig. 2h,i). Eventually all peptides will be hydrolyzed to form amino acids.
From the above discussion, we can conclude that so long as the hydrolytic process is controlled to the extent that there is a small amount of intact protein remaining in the hydrolysate, the hydrolysis rate of a terminal peptide to form internal peptides is always much smaller than that of the intact protein. As a result, the terminal peptides will be the dominant components in the final solution along with the intact protein. Owing to the limited detection dynamic range of the mass spectrometer and the ion signal suppression effect in MALDI, direct MALDI analysis of the hydrolysate only allows the detection of the intact protein and the terminal peptides. Signals from the low-abundance internal peptides are suppressed and not seen in the MALDI spectrum. As a result, peptide peaks detected in the spectrum are exclusively from the terminal peptides. We note that, to account more accurately for the acid hydrolysis process, we are currently in the process of determining hydrolysis reaction constants of proteins under various conditions with the goal of developing a quantitative description of the hydrolysis kinetics involved in the MAP sequencing technique. Our initial quantitative model is presented in the Supplementary Note online.
Using the properly controlled hydrolysis conditions (Fig. 2), the MAP sequencing technique is found to be generally applicable to a wide range of proteins, including proteins containing internal disulfide bonds and proteins containing acid labile bonds such as aspartic acid (D)-proline (P). For proteins containing internal disulfide bonds, protein reduction is required before MAP sequencing. This is illustrated in Supplementary Figure 2 online where lysozyme (129 amino acids and MW 14,307 Da) was reduced to different extents and then subjected to MAP sequencing. For this protein, polypeptide ladders covering 100% of the protein sequence were obtained from one experiment.
Because the success of the MAP sequencing technique depends on near-uniform hydrolysis of all peptide bonds in a protein, it would appear to be difficult to apply this technique to sequencing proteins containing acid labile bonds such as D-P. It turns out that this technique can be readily applied to sequencing these proteins. This is shown in Figure 3a−c where a protein containing a D-P bond isolated from an E. coli extract was subjected to MAP sequencing. Apparently this high-performance liquid chromatography (HPLC)-fractionated sample contains some low-mass peptide and protein impurities. The MALDI spectrum of the sample and the expanded spectra displaying impurity peaks in the low-mass region are shown in Supplementary Figure 3 online. Panels a and b of Figure 3 show the MALDI spectra obtained from the sample after acid hydrolysis with microwave irradiation for 30 and 60 s, respectively. A number of polypeptide peaks are observed, including one intense peak arising from the N-terminal peptide generated from the breakage of the D-P bond of the protein identified as YMDF-ECOLI (P56614). The corresponding C-terminal peptide is also observed. With the increase in irradiation time from 30 to 60 s, the signal-to-noise ratios of the polypeptide peaks are improved. However, the relative intensities calculated from the peak areas between other polypeptides and the two peptides from the D-P bond breakage remain similar. The expanded spectrum of Figure 3b is shown in Figure 3c where the entire sequence of the protein can be read from the N- and C-terminal ladders. Three peaks labeled with 'I' are the possible internal fragments from the follow-up hydrolysis of the two terminal peptides generated from the D-P bond breakage. Comparison of the spectra shown in Figure 3c to the expanded spectra shown in Supplementary Figure 3 online indicates that several other peaks labeled as 'X' in Figure 3c are from the impurities present in the sample. These impurities have much lower concentrations than that of the main protein in the original sample. Thus they did not hydrolyze extensively. More importantly, the presence of the impurity peaks did not interfere with the assignment of the terminal peptide ladders of the main protein. Another example of MAP sequencing of proteins containing D-P bonds (that is, RL33-ECOLI (P02436) isolated from E. coli) is given in Supplementary Figure 4 online. It is clear that the presence of acid labile bonds, such as D-P, in a protein does not prevent the generation of a complete set of terminal ladders.
| | |
The detection sensitivity of the MAP technique was also examined. For small proteins with molecular masses of up to about 14,000 Da, the sample required for the experiment is generally less than 1 pmol. Lower amounts of sample can be used, but as the amount decreases, the polypeptide peaks in the high-mass region start to decrease (Supplementary Fig. 5 online). This level of detection is consistent with the current practice of using a microliter sample deposition method where small proteins and large peptides can be detected at 1 fmol using MALDI-time-of-flight (TOF) analysis17. With nanoliter sample deposition, MALDI sensitivity can be improved by 100-fold or more18,
19. Thus, future work on miniaturizing the hydrolysis process followed by nanoliter sample deposition should substantially improve the overall sensitivity of the MAP technique.
With the generation of polypeptide ladders, information on sequence and post-translational modifications can be deduced from the mass spectra. The mass difference between adjacent peaks of the same series of polypeptides corresponds to the mass of an amino acid residue, which forms the basis for its identification as well as its modification, if any (Fig. 1). Acid labile modifications may be destroyed during the hydrolysis; but they are rare20. Among the twenty common amino acids, leucine and isoleucine have the same mass and, therefore, cannot be distinguished by this method. Glutamine and lysine have similar masses and cannot be readily distinguished within the molecular mass measurement accuracy of TOF MS; but it should be entirely possible with MALDI Fourier Transform-ion cyclotron resonance (FT-ICR) MS21. However, distinguishing these pairs of amino acids is only required for de novo sequencing of an unknown protein from a species with no or little genome or proteome database. Fortunately, with the rapid expansion of genome databases as well as the possibility of performing cross-species database searching, de novo sequencing is becoming an increasingly rare practice. Thus the MAP sequencing technique should be very powerful for reexamining protein sequences translated from their genome and determining their modifications.
As an example, the expanded MALDI spectra of cytochrome c after 60-s microwave irradiation in HCl are shown in Figure 3d. A long stretch of sequence can be deduced from a ladder and the sequences read from two ladders have an overlap. The combination of the two allows the determination of the entire sequence of cytochrome c with information on possible modifications. For example, the mass difference between the molecular ion peak and the peak at m/z 12,147 is 214 Da, which matches the mass of acetylated glycine linked to D. Another modification found in cytochrome c is the heme group covalently bound to cysteines 14 and 17. In this case, polypeptide ladders from 1−17 to 1−104 all have a positive mass shift corresponding to the mass of heme. Polypeptides 1−13, 1−12 and others, do not have the mass shift. Furthermore, because of the heme attachment to cysteines 14 and 17, which apparently hinders the hydrolysis of internal peptide bonds, polypeptide peaks generated from the hydrolysis of peptide bonds between these two residues were not detected. Taken together the evidence confirms the heme modification and its location on cysteines 14 and 17 in cytochrome c.
One of the anticipated major applications of the MAP sequencing technique is to determine the phosphorylation sites of a phosphorylated protein. As an example, an  -casein sample and a dephosphorylated -casein sample were subjected to MAP sequencing (Fig. 4). Because both samples contain about 15% of -S2-casein, there were a number of low intensity peaks (some are labeled in the figure) in addition to the more intense polypeptide peaks generated from -S1-casein. Despite the presence of these low intensity peaks, the major peaks were readily assigned to the sequence of -S1-casein. In addition, mass shifts corresponding to one or more phosphate groups in 80-Da increments were identified, which in turn provided the information for identification of the phosphorylation sites. Determining the modification site is easy and unambiguous, because all polypeptides in a ladder containing one modification will have the 80-Da mass shift until they encounter another modification which will increase the mass by another 80 Da. In this case, the polypeptide ladders from N-terminal 16−31 to 16−84 and C-terminal 137−214 to 198−214 were detected, resulting in the survey of 114 amino acids. In addition, mass analysis of terminal peptides 16−31 and 198−214 indicated that there were no modifications on these peptides. Thus a total of 145 amino acids were examined. In the protein sequence covered by these amino acids, there are six known phosphorylation sites. The MAP sequencing technique (Fig. 4) detected all six sites in one experiment. The internal sequence from residue 85 to 136 was not covered in this experiment because of the suppression of high-mass peaks (>9,000 Da) in MALDI. One obvious approach to map this portion of the sequence would use chemical or limited enzyme digestion of the protein to generate large peptides, followed by HPLC separation and MAP sequencing. However, we believe that technical advances in the near future such as using more powerful mass spectrometers21,
22,
23 and better sample preparation24,
25 should allow direct sequencing of a protein of this size (see below).
| | |
Although the MAP sequencing technique requires a relatively pure protein for unambiguous amino acid sequencing and determination of chemical modifications, the above example also illustrates that sequence and modification information can still be obtained even when about 15% of the sample are other components or impurities. The applicability of the technique for sequencing a protein containing impurities clearly depends on the type of information to be generated and the nature of the impurities, that is, whether the impurities will interfere with the assignment of the sequence ladders from the protein of interest. The impurity peaks shown in Figure 3a−c did not interfere with the reading of the terminal ladders, and the protein was completely sequenced. In applying the MAP sequencing technique to real world samples, another important consideration is whether this technique can tolerate common additives, such as surfactants. SDS is perhaps the most difficult surfactant to deal with in the mass spectrometric analysis of proteins26. However, recent advances in sample preparation methods, such as the use of a two-layer method, allow direct analysis of protein and peptide samples containing a small amount of SDS, albeit with reduced sensitivity27,
28. We have investigated the effect of SDS on acid hydrolysis with microwave irradiation (see Supplementary Fig. 6 online). We found that the effect of SDS on acid hydrolysis is small for samples containing <0.1% SDS. Thus the technique appears to provide moderate tolerance to SDS, which should prove to be important in situations where the presence of SDS in a sample is unavoidable, such as in dealing with membrane proteins.
Because of its high sensitivity, speed and specificity, the MAP sequencing technique should be very useful for detailed characterization of proteins on a large scale. Proteins electroeluted or extracted out of a gel or isolated by HPLC can be directly sequenced. As an example, a protein extract from E. coli K12 was fractionated using HPLC. A total of 28 proteins within the mass range accessible to complete sequencing by the current MAP technique were subjected to sequencing (Supplementary Table 1 online). Half of the 28 proteins were hypothetical proteins or unknown proteins as indicated in the Swiss-Prot and NCBI proteome database. In all cases, the presence or absence of the methionine start codon in the expressed proteins was unambiguously determined. As well, loss of signal peptides for a number of proteins was positively identified. In some cases, methionine oxidation or disulfide bond formation was observed. These results demonstrate that the MAP sequencing technique has the potential for rapid mapping of a large number of proteins from cells, tissues and other sources, as well as for production monitoring and quality control of recombinant proteins.
Finally, it should be noted that future improvements in mass spectrometric instrumentation and sample handling methodology should expand the applicability of the MAP technique to large proteins. The examples given here show that, for the current MAP technique, there is an upper mass limit for detecting polypeptide peaks. The useful mass region is generally limited to below 14,000 Da. This limit most likely results from the problem associated with peptide detection, not the hydrolysis process itself. The peak intensity decreases as the polypeptide mass increases, which is expected in MALDI-TOF where the ionization efficiency drops as the analyte mass increases17 (Fig. 2d). Detector saturation also plays some role25. We note that analyzing polypeptide ladders is analogous to analyzing an industrial polymer, such as polystyrene, with a broad oligomer distribution where high-mass oligomers are usually not detected24,
25. Analyzing a polydisperse polymer can be done using size-exclusion chromatography to prefractionate the sample into several polymers of narrow polydiversity, followed by MALDI analysis of individual fractions29. Similarly we believe that the upper mass limit of the current MAP technique can be extended by using chromatography to mass-fractionate polypeptides after hydrolysis and then analyze the individual fractions by MALDI. Ultimately the mass limit is likely to be imposed by the mass resolution required to resolve adjacent peptides and mass measurement accuracy. Resolution requirements may be relaxed by designing experiments to fractionate N- and C-terminal peptides into two groups (e.g., using an affinity tag at the terminus combined with affinity purification), followed by MAP sequencing of the two fractions. The use of orthogonal MALDI-TOF22,
23, which provides better resolution and accuracy than the conventional MALDI-TOF used in this work, and MALDI FT-ICR21, which is superior to MALDI-TOF in terms of mass resolution and mass measurement accuracy, may greatly extend the useful mass range of the MAP sequencing technique, allowing the sequencing of large proteins.
In summary, during the course of our investigation of using acid hydrolysis as a means of generating small peptides, we discovered that proteins could be readily hydrolyzed after a brief exposure to microwave irradiation to form predominately two series of polypeptide ladders: one containing the N-terminal amino acid and another one containing the C-terminal amino acid. MALDI analysis of the hydrolysate produced a simple mass spectrum consisting of peaks from the N- and C-terminal peptide ladders exclusively. Mass analysis of the polypeptide ladders allowed rapid determination of protein sequences and modifications.
Methods
MAP sequencing experiment.
A microliter of protein sample was mixed with an equal volume of 6M HCl in a 0.6-ml polypropylene vial. The vial was capped and then placed inside a household microwave oven with 900 W output at 2,450 MHz. A container with 100 ml of water was placed beside the sample vial to absorb the extra microwave energy. The microwave oven was turned on for, typically, 60 s. After <2 min of microwave irradiation, the bottom of the vial was found to be slightly warm. The temperature of the solution inside the vial was unknown; but no visible boiling or depletion of the solution was noted. After microwave irradiation, the sample vial was taken out of the microwave oven and the solution in the vial was dried under vacuum centrifugation. The dried sample was redissolved in a matrix solution of -cyano-4-hydroxycinnamic acid. The mixture was then deposited on a sample target using a two-layer sample preparation method30 for MALDI analysis. MALDI MS experiments were carried out on a Bruker Reflex III TOF mass spectrometer using a linear mode of operation. Ionization was performed with a 337-nm pulsed nitrogen laser.
E. coli K12 protein analysis experiment.
Proteins were extracted from the cells by using 0.1% trifluoroacetic acid (TFA). Proteins were first separated by strong cation ion exchange chromatography (Vydac 400VHP 81, 1 mm internal diameter (i.d.)  150 mm) using an Agilent 1100 HPLC system. A linear gradient was used in 60 min (A, 20% acetonitrile and 0.1% TFA in water; B, 1 M NaCl in A). The eluates were fractionated by chromatographic peaks. Individual fractions were further separated by reversed-phase HPLC (C8, Vydac, 1 mm i.d. 150 mm) using an Agilent 1100 capillary HPLC system. A linear gradient was used in 60 min (A, 0.1% TFA in water; B, 0.1% TFA in acetonitrile). The eluates were fractionated by protein peaks detected in the UV chromatogram.
Note: Supplementary information is available on the Nature Biotechnology website.
Received 14 April 2004; Accepted 23 July 2004; Published online: 7 September 2004.
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Acknowledgments
This work was supported by the Natural Sciences and Engineering Research Council of Canada, Genome Canada through Genome Prairie's Enabling Technologies Project, Canada Foundation for Innovation, The Protein Engineering Network of Centres of Excellence, and Alberta Cancer Board. We thank Andrew Shaw (Cross Cancer Institute, Alberta) for helpful comments on the manuscript.
Competing interests statement:
The authors declare that they have no competing financial interests. |
) with a solid underline) and from the C-terminal ladder ((
) with a dashed underline), of YMDF-ECOLI (P56614) and cytochrome c, respectively.
-S1-casein and MALDI spectrum (in black) of a sample containing 85% dephosphorylated 
