Native Protein Proteolysis in an Immobilized Enzyme Reactor as a

Article pubs.acs.org/ac

Native Protein Proteolysis in an Immobilized Enzyme Reactor as a Function of Temperature Dinelia Rivera-Burgos and Fred E. Regnier* Department of Chemistry, 560 Oval Drive, Purdue University, West Lafayette, Indiana 47906, United States ABSTRACT: Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis.

A

signature peptide products is unknown and not stoichiometrically related to protein concentration. A second complicating factor in proteolysis is that proteins are highly structured. Many of the requisite cleavage sites for peptide generation are buried within the rigid three-dimensional structure of proteins. Although trypsin digestion is facilitated by disrupting protein structure through reduction of disulfide bonds and addition of urea or guanidinium hydrochloride,5 there is no evidence these proteolysis facilitators globally unmask the peptide bonds required for trypsin proteolysis. Clearly cleavage sites within proteins are being unmasked during the course of proteolysis. This is an important issue because at high concentrations of trypsin the unmasking of lysine and arginine cleavage sites could be rate limiting with rigid proteins. Recent studies with immobilized trypsin reactors have shown that the incidence of miscleaved peptides is more likely from some regions of a protein than others.6 The interpretation of these results was that miscleaved fragments

lthough the core of proteomics is determining the molecular weight of protein fragments along with correlating peptide and gene sequence, an important prerequisite in the bottom-up strategy is converting proteins to peptide fragments that are amenable to mass spectral analysis.1 Generation of these peptides with trypsin is generally explained by a model in which a protein is stoichiometrically converted to a mixture of peptides having single lysine or arginine residues located at their C-terminus,2 referred to herein as “limit peptides”. But this may not always be the case. As an endopeptidase, trypsin is inclined not to generate limit peptides;3 instead, it tends initially to form fragments bearing multiple lysine or arginine residues that are often referred to as “miscleaved tryptic peptides”. Conversion of these miscleaved peptides to limit peptides requires trypsin to switch from being an endopeptidase to an exopeptidase. This is more likely to occur in the later stages of proteolysis,4 causing trypsin to slow down as digestion progresses and diminishing conversion of miscleaved peptides to limit peptides. This has important ramifications in protein quantification. Limit peptides will not always appear as equimolar surrogates of a protein. When multiple peptides of the same partial sequence are formed during proteolysis, the molar ratio between the parent and © 2012 American Chemical Society

Received: April 27, 2012 Accepted: July 16, 2012 Published: July 16, 2012 7021

dx.doi.org/10.1021/ac301114m | Anal. Chem. 2012, 84, 7021−7028

Analytical Chemistry

Article

50 mm2 IMER operated at fixed temperature in the flow mode (100 μL/min). The digestion time was 4 min in all cases. Four different temperatures were used: 27, 37, 45, and 60 °C. Reduced and alkylated proteins were examined at 37 °C while native proteins were studied at all four temperatures. Digested transferrin fractions collected from the IMER were further fractionated by RPC and collected in 96 fractions that were analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI) using α-cyano-4-hydroxy-cinnamic acid (CHCA) ionization matrix. Use of CHCA is significant because it reduces disulfide-coupled peptides to individual, reduced peptides. In the case of the small number of proteins from the yeast lysate, the peptide mixtures resulting from the IMER proteolysis were fractionated by RPC where the HPLC system was coupled directly to a LTQ Orbitrap hybrid mass spectrometer. Both MALDI and Orbitrap data were analyzed by Protein Pilot 2.0 software as described below. Immobilized Trypsin Digestion. For denaturation, reduction, and alkylation, vacuum-dried protein samples were reconstituted with 8 M urea in 50 mM TRIS buffer containing 10 mM CaCl2 at pH 8.0. Proteins thus denatured were reduced with 10 mM DTT. After 1 h incubation at 60 °C, iodoacetic acid was added to a final concentration of 20 mM for alkylation and incubated in darkness for 30 min at room temperature. A final concentration of 1 M urea was obtained after dilution with 50 mM TRIS buffer containing 10 mM CaCl2 (pH 8.0). For nonreduced and nonalkylated proteolysis, the vacuum-dried protein samples were just dissolved in DI water. A 100 μL (500 ng/mL) aliquot was automatically injected into the Perfinity Workstation (Perfinity Biosciences, West Lafayette, IN) equipped with a 4.6 × 50 mm2 Perfinity IMER. Elution was obtained with a flow rate of 100 μL/min for a total reaction time of 4 min. A single reference sample of reduced and alkylated transferrin and yeast lysate was digested with immobilized trypsin at 37 °C, whereas proteolysis of native human transferrin and yeast was achieved at room temperature (RT) and 37, 45, and 60 °C using the same IMER and optimized digestion buffer to maintain the reactor pH and elute peptide digests. Reversed Phase Chromatography Fractionation of Tryptic Digests. Tryptic digests generated after the proteolysis of transferrin were fractionated by reversed phase chromatography (RPC) using a Pepmap C18 nanocolumn [3.5 μm particle size Zorbax 300sB-C18 packed in 100 μm × 15 cm glass column (Agilent Technologies, Santa Clara, CA)] and an Agilent 1100 nanoflow HPLC. The RPC separation was achieved using a 70 min linear gradient from 97% solvent A with 3% solvent B to 70% solvent A and 30% solvent B at a flow rate of 800 nL/min. Solvent A was composed of DI water to which trifluoroacetic acid (TFA) had been added to a concentration of 0.1%. Solvent B was prepared with HPLC grade acetonitrile (ACN) to which TFA had been added to a concentration of 0.1%. Peptide fractions from the RPC column were continuously mixed with MALDI matrix (α-cyano-4hydroxycinnamic acid, 4 mg/mL in 60% ACN, 40% H2O, 0.1% TFA) using a mixing tee at the end of the RPC column at a flow rate of 1.2 μL/min. A total of 96 fractions were collected directly onto a 384 target, stainless steel MALDI plate utilizing a microfraction collector, and MALDI spotter driven by the Agilent 1100 HPLC instrument. MALDI-TOF/TOF-Based Characterization of Peptides. An ABI model 4800 MALDI-TOF/TOF proteomics analyzer equipped with a 200 Hz Nd:Yag laser was used in the positive

arise from regions in proteins where peptide bonds are shielded and become available for cleavage later in proteolysis than those available initially on the surface of proteins. As a consequence, they are less likely to be completely digested, leaving miscleaved peptides at the end of proteolysis. This phenomenon further impacts the quantification issue noted above. It is clear that trypsin concentration and the unmasking of cleavage sites play important roles in product stoichiometry and the number of limit peptides generated during proteolysis. The work reported here describes studies on protein proteolysis in which (i) reaction temperature was elevated to enhance the unmasking of cleavage sites, (ii) trypsin concentration was increased beyond that normally used in solution digestion by using an immobilized enzyme reactor to facilitate limit peptide generation, and (iii) native proteins were digested. Use of the term “native proteins” throughout this paper is meant to designate sample origin and absence of chemical modification before proteolysis, not that the proteins are of native conformation. It is highly unlikely that proteins will be of native structure at either elevated temperatures or subsequent to initial peptide bond cleavage. The hypothesis being examined was that limit peptides could be rapidly obtained from native proteins without reduction, alkylation, and the use of high urea concentrations and surfactants. Transferrin was chosen as a model protein because of its known resistance to trypsin digestion;7 in addition, a series of yeast proteins were examined as well. A commercial IMER was used that the manufacturer claims has at least 100 times higher trypsin concentration than the 50:1 protein:trypsin ratio used in solution based proteolysis.

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EXPERIMENTAL SECTION Materials and Chemicals Reagents. DL-Dithiothreitol (DTT), iodoacetic acid (IAA), urea, N-tosyl-L-lysyl chloromethyl ketone (TLCK), HPLC grade acetonitrile (ACN), trifluoroacetic acid (TFA), α-cyano,4-hydroxy-cinnamic acid (CHCA), and human apo-transferrin were purchased from Sigma Aldrich (St. Louis, MO). Tris(hydroxymethyl)-aminomethane (TRIS) was obtained from Bio-Rad (Hercules, CA). Calcium chloride was purchased from J.T Baker (Philipsburg, NJ). Trypsin gold, mass spectrometry grade enzyme, was acquired from Promega Corporation (Madison, WI). The ABI 4800 proteomics analyzer calibration mixture (4800 Cal Mix, bradykinin, angiotensin I, glu-fibrinopeptide B, ACTH fragment 1−17, ACTH fragment 18−39, and ACTH fragment 7− 38) was purchased from Applied Biosystems (ABI, Foster City, CA). The immobilized enzyme reactor (IMER) and the Perfinity optimized digestion buffer were obtained from Perfinitiy Biosciences (West Lafayette, IN). The Perfinity Workstation was from Perfinity Biosciences. Experimental Design. The objective of the work described here was to determine the difference in the efficiency of proteolysis between native and reduced proteins at high levels of trypsin and elevated temperature. Two types of samples were examined; the model protein transferrin and a small number of proteins from a yeast lysate. The yeast proteins were chosen on the basis of differences in structure and characteristics such as molecular size and number of disulfide bonds. The transferrin sample was examined by MALDI-MS while the yeast proteins were studied by ESI-MS. The studies described here were executed using two forms of the protein samples: the native protein and the reduced and alkylated form. Proteins were introduced directly into a 4.6 × 7022



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Figure 1. Peptides generated by trypsin-IMER digestion of native and reduced transferrin.

was coupled directly to the LTQ Orbitrap hybrid mass spectrometer (Thermoelectron, San Jose, CA). The LTQ Orbitrap was equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The MS was operated in the data-dependent mode, in which a survey full scan MS spectrum (from m/z 300 to 1600) was acquired in the Orbitrap with a resolution of 60 000 at m/z 400. This was then followed by MS/MS scans of the 3 most abundant ions with +2 to +3 charge states. The resulting fragment ions were recorded in the linear ion trap. Mass Spectrometry Data Processing. Protein identifications based on acquired MS/MS spectra were carried out using Protein Pilot software 2.0 with the Pro Group algorithm for protein identification. The minimum acceptance criterion

ion mode for the analysis of peptides spotted onto the MALDI plate. The 4000 Explorer software furnished with the ABI 4800 allowed automated acquisition of MS and MS/MS data. LTQ Orbitrap XL-Based Protein Characterization. The peptide mixtures resulting from the IMER proteolysis of reduced and alkylated as well as native yeast extract were separated on an Agilent 1100 HPLC system using a 75 μm × 120 mm C18 reversed phase chromatography (RPC) column packed with 5 μm C18 Magic beads. Peptide separations were achieved using an 85 min linear mobile phase gradient from 0.1% formic acid in water to 0.1% formic acid in acetonitrile at a flow rate of 0.3 μL/min. The electrospray ionization emitter tip was generated on the prepacked column with a laser puller (Model P-2000, Sutter Instrument Co.). The HPLC system 7023



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An interesting feature of the high frequency cleavage sites is that in some cases large numbers of peptides were formed both C-terminal and N-terminal to the cleavage site while in other cases peptides were formed in only one direction from the cleavage sites. Cleavage at sites K618 and R642 in reduced transferrin provide examples. Three peptides are formed with K618 as the C-terminal amino acid while no peptides were formed on the N-terminal side of this cleavage site. At residue R642 in contrast, peptides were formed on both sides of the cleavage site. Three peptides were generated in which R642 was the C-terminal residue while four were formed with S643 at the N-terminus. The same general phenomenon was seen with native transferrin, but at different sites. Moreover, most of the peptides derived from these high frequency sites were miscleaved peptides. These results are interpreted to mean that high frequency cleavage sites are points of initial cleavage within a region. Subsequent to initial cleavage, portions of the primary structure from this point are further digested. Trypsin engages these sites primarily as an endopeptidase, generating predominantly miscleaved peptides (Figure 2). On the basis of the fact that

for peptide identification was the 95% confidence level. Proteins were identified on the basis of the presence of at least two unmodified peptides from the same protein identified by the Pro Group algorithm at the 95% confidence level. An unused score cutoff of 4 was the minimum value for identifying proteins with the Protein Pilot 2.0 software.

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RESULTS Presentation of Data. A summary of the results from the five transferrin samples is presented in Figure 1 where the data is organized around the linear sequence of transferrin, beginning on the left from the N-terminus. The series of horizontal brackets above and below the sequence line indicate peptides that were identified in digests. Those in red brackets above the sequence line were derived from native transferrin while the black brackets below were obtained from reduced transferrin exclusively. Brackets above the sequence line in purple indicate peptides that were found in both native and reduced transferrin while peptides bracketed by red lines were obtained exclusively from native transferrin. The temperature at which a peptide was obtained is designated within the bracketing line. Dotted blue brackets above the sequence line indicate limit peptides that were derived from native transferrin. Those with a heavier dotted blue and red line designate limit peptides of equimolar concentration to their protein parent; i.e., no other peptide bears any of the sequence in these peptides. This means these peptides could be used in stable isotope based internal standard quantification. The vertical red arrows above the sequence line indicate cleavage sites in the native protein from which three or more peptides were produced Nterminal or C-terminal to the point of hydrolysis. These sites are referred to as “high frequency cleavage sites” in the discussion that follows. Vertical black arrows below the sequence line indicate preferred cleavage sites for the reduced protein. Green arrows appearing at an angle below the sequence line indicate points of hydrolysis in reduced transferrin not involving a lysine or arginine residue. Angled purple arrows above the sequence line show similar sites of cleavage in native transferrin at amino acids other than lysine or arginine. The large vertical yellow arrows at asparagine residues are glycosylation sites. Frequent Cleavage Sites. Clearly IMER based proteolysis of native and reduced transferrin is quite different as seen in Figure 1. One is in the occurrence of cleavage sites from which three of more peptides bearing the same amino acid at their Nor C-terminus were formed. The 10 high frequency cleavage sites are noted by the red arrows with native transferrin at R26, R143, K225, R251, R273, K310, R475, R541, K553, and K571 while five noted by black arrows were seen with reduced transferrin at K37, K60, K163, K618, and R642. These results are interpreted to mean these sites bear unmasked lysine or arginine residues and are available for immediate attack by trypsin. The very large difference in the location of these between the two forms of transferrin suggests substantial conformational dissimilarity. It is particularly significant that with native transferrin some of these sites only emerged at 45−60 °C. This is probably in association with conformational changes in transferrin,8 not from increases in the rate of proteolysis at elevated temperature. The close proximity of sites R541, K553, and K571 along with K225, R251, and R273 in native transferrin suggests they are in unmasked regions. The same is perhaps true in reduced transferrin at sites K37 and K60 and those in the region represented by K618 and R642.

Figure 2. Formation of limit tryptic peptides requires trypsin to act as both an endopeptidase and exopeptidase. Peptides appearing in blue are limit tryptic peptides.

most of the miscleaved peptides observed in this work have no more than two missed cleavage sites, most subsequent digestion of peptides by trypsin will be as an exopeptidase. Given enough time all miscleaved peptides will be digested to limit peptides, but reaction time is determined by residence time in the IMER. Limit Peptides. As noted above, the precedent in proteomics is to identify and quantify proteins through limit peptides. However, as seen in Figure 1 many of the products formed are miscleaved peptides that must be converted into limit peptides. This probably occurs in multiple ways (Figure 2), either (i) in a second step after initial cleavage of the protein backbone while the peptide is still attached to a major section of the protein or (ii) in a later step during the digestion of miscleaved tryptic peptides. The second of these options is the most likely on the basis of the propensity of trypsin to act as an endopeptidase. Limit peptides are identified in Figure 1 with two types of markings. When marked with blue brackets having a single dotted line, the limit peptide was found in native transferrin digests along with other miscleaved peptides bearing part of the same sequence. This was true of 22 limit peptides from native transferrin. The second case was of limit peptides that appeared in native transferrin digests alone, with no other peptides bearing any portion of their sequence. These peptides are identified with blue brackets connected with two dotted blue lines and a red dotted line. Five limit peptides were found that met this criterion. Because no part of their sequence is shared with another peptide they are equimolar in concentration to their parent protein and can be used in protein quantification. 7024



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Temperature. Elevating the temperature of proteolysis had a dramatic effect on peptide formation, both in terms of the degree to which the entire sequence of the protein was observed by tryptic peptides and the number of limit peptides (Figure 3). At room temperature approximately 28% of the

production should gradually accelerate with temperature, not appear suddenly with an 8 °C change in temperature. Glycosylation Sites. Transferrin has two glycosylation sites; one at N432 and another at N630. The site at N432 is totally occupied in all cases while the site at N630 is only partially occupied. Peptides carrying residue N432 were not seen in the case of either native or reduced transferrin. This is to be expected in view of the fact that glycopeptides do not ionize well with the α-cyano-4-hydroxy-cinnamic acid (CHCA) MALDI matrix used in these studies. The fact that a peptide bearing N630 was seen is attributed to the fact this site is not fully glycosylated, but it was only seen in the case of reduced transferrin. Disulfide Bonds. Native transferrin has 20 disulfide bonds along with bound iron. As observed in the data above, the native and reduced forms would be expected to behave very differently during trypsin digestion based on disulfides reducing peptide bond demasking. This was certainly true in terms of peptide sequence and the number of peptides obtained at 27 °C, but at 45 and 60 °C more peptides were formed. Native transferrin produced roughly the same number of peptides as the reduced and alkylated form at elevated temperature (Figure 4). Five exclusive limit peptides were even obtained from native transferrin, among them being SAGWNIPIGLLYCDLPEPR, WCLSHHER, and DKEACVHKILR. These three peptides are involved in disulfide bridges but were seen as being reduced by MALDI-MS. This brought up the question of how disulfide reduction could occur with native proteins which had no contact with reducing agents during proteolysis. Proteolysis of native proteins with disulfide bridges produces dipeptides with the peptides coupled through a disulfide linkage,9 although some scrambling could occur with storage at pH 8.0. This linkage is stable during reversed phase chromatography, but disulfide bond reduction can occur during ionization with α-cyano-4-hydroxy-cinnamic acid.10,11 Clearly, that occurred in this case. Similar results were seen in the electrospray mode of ionization with a series of yeast proteins (Table 1). Coverage was slightly higher in nonreduced and alkylated proteins at 37 °C in 7 of the 13 proteins examined without regard to the number of disulfides. At 45−60 °C coverage was higher without reduction and alkylation in 12 of the 13 proteins. Disulfide content reduced coverage at 27 °C relative to reduced and alkylated forms, but this difference was easily overcome at 45− 60 °C as seen with heat shock proteins SSA2 (1 disulfide), pyruvate kinase (3 disulfides), and adenosylhomocysteinase (5 disulfides). The most difficult proteins to digest were the 40S ribosomal proteins S18−B and S15, neither of which bore disulfides. Clearly disulfide content had minimal impact on coverage in either the MALDI or ESI modes of ionization, especially when IMER temperature is elevated to 45 °C or higher. Although different forms of sample treatment may change the blend of peptides obtained upon proteolysis, it seems to have minimal impact on coverage with the trypsin IMER used in these studies. Nontryptic Cleavages. Trypsin also cleaves proteins with low frequency at sites other than lysine and arginine. The mechanism by which this occurs has never been well explained. The only thing noteworthy in this study was that more nontryptic cleavages were seen with reduced than native transferrin and they were at different sites.

Figure 3. Impact of IMER temperature on proteolysis.

structure of native transferrin was observed through tryptic peptides. This increased to slightly over 70% at 60 °C. Reduced transferrin in contrast showed 84% coverage at room temperature, much higher. Clearly cleavage sites in reduced transferrin are less shielded than in native transferrin at room temperature. The number of limit peptides also increased with temperature; showing approximately twice the number at 60 °C as at room temperature. It is seen in Figure 4 that 10 of the peptides observed at 60 °C were seen at all the temperatures studied while two of the peptides present at room temperature and 37 °C were absent at 45 and 60 °C. The fact that many of these peptides only appear at elevated temperature and were not degradation products of peptides with miscleavages is interpreted to mean that they arose from temperature initiated alterations in protein structure, not from temperature accelerated rates of proteolysis. This comment is based on the fact that the rate of proteolysis and peptide

Figure 4. Venn diagram showing regions where limit tryptic peptides were generated after the IMER digestion at different temperatures of the native form of transferrin. 7025



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Table 1. ESI-MS Analysis of Multiple Proteins from Yeast Lysate: Percent of Coverage as a Function of Temperature between Native and Reduced Proteins protein identification pyruvate kinase 1 heat shock protein SSA2 phosphoglycerate kinase adenosylhomocysteinase 60S ribosomal protein L3 (DL)-glycerol-3-phosphatase 1 40S ribosomal protein S15 alcohol dehydrogenase 1 40S ribosomal protein S18−B eukaryotic translation initiation factor 5 inorganic pyrophosphatase translationally controlled tumor protein homologue peptidyl-prolyl cis−trans isomerase

no. Cys residues

no. disulfide bonds

reduced and alkylated yeast 37 °C (%)

native yeast 27 °C (%)




7 3 0 11 0 3 0 8 0 7

3 1 0 5 0 1 0 4 0 3

44.60 38.34 53.61 21.83 10.34 34.00 9.16 30.46 45.21 11.46

29.00 23.16 37.50 13.81 22.57 26.40 16.90 6.03 10.27 6.42

35.00 43.19 63.22 18.49 33.00 45.60 24.65 20.69 17.81 22.29

43.60 45.54 68.03 21.83 33.00 54.80 24.65 30.75 20.55 22.29

53.60 43.51 79.09 26.73 47.79 55.20 24.65 33.05 15.07 22.29

0 0

0 0

32.75 23.35

8.36 9.58

19.86 22.16

49.83 26.35

34.84 34.84

2

1

24.07

9.26

33.95

60.49

60.49

Figure 5. Hypothetical mechanism of protein proteolysis. “X” designates a basic amino acid in the polypeptide backbone of the protein. Illustration A shows a point of initial cleavage in a section of the polypeptide backbone that is unmasked and slightly away from the protein surface. Subsequent to the initial cleavage a portion of the polypeptide either C-terminal or N-terminal to the initial cleavage site is illustrated as lifting up from the surface and becoming available for secondary cleavage. The illustration in B shows N-terminal piece still adsorbed to the surface while the C-terminal portion has lifted from the surface. The illustration in C shows both parts of the cleave backbone lifting from the surface.

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DISCUSSION Mechanism of Proteolysis. The question of how trypsin digests a protein like transferrin is intriguing. Where does proteolysis start, and how does it proceed? The temperature at which proteolysis occurs plays a role as well. Clearly raising the temperature to 45 °C altered protein structure in most cases. With 79 basic amino acids there are a large number of places in transferrin where proteolysis could theoretically start. One option would be that proteolysis could be completely random; i.e. digestions could start at any one of the 26 arginine or 53 lysine residues. A second possibility would be that it could be more directed, as by some structural feature(s). Going on to the second step, where does the second cleavage occur that releases a peptide from the parent structure? The second cleavage site could again be selected at random or be directed in some way. With the random model of proteolysis the molecular weight of the fragments would gradually decline in a cascading series of steps to the level of limit peptides. The

targeted model in contrast would suggest initial cleavage at a smaller number of sites followed by directed second steps. Fragment molecular weight in directed proteolysis would tend to be smaller than with random proteolysis. Data from the studies reported here suggest an intermediate model of proteolysis. Proteolysis is neither completely random nor totally directed. Proteolysis seems to start at a small number of preferred cleavage sites, ranging from 5 to 10 in the initial stages of transferrin proteolysis selected from among 79 basic amino acid residues. Because cleavage at these high frequency sites was totally different for native and reduced transferrin, it is concluded that conformational differences arising from disulfide bridges in the native protein are responsible for the difference. The same was true with temperature. Elevating the reaction temperature altered the amount of cleavage at some sites more than others. The most probable explanation is that these preferred sites are on the surface of the protein. A second criterion would be that they 7026



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less than 10 min with an immobilized trypsin reactor. The great advantage of using an IMER to carry out proteolysis is that the amount of trypsin per unit of proteins is so high the reaction is still completed in reasonable time.

have to be sufficiently free to move away from the protein structure far enough to enter the active site of trypsin (Figure 5). As we know from affinity chromatography, substrates attached to a surface have to be 10−15 Å away from the surface to effectively enter the active site of most enzymes. The same would be true in proteolysis. A basic amino acid residue could be on the surface of a protein but is still strongly enough associated with adjacent amino acids to preclude availability for binding to trypsin. Having made the 4−5 initial cleavages, what happens next? Portions of the primary structure in an N- or C-terminal direction from the initial cleavage site can either move away or still stay associated with the protein on the basis of forces involved in forming the 3-D structure prior to proteolysis. In either case, the initial cleavage fragment will still have substantial structure. We speculate that after the initial cleavage a portion of the polypeptide backbone can lift up from the surface and become available for further proteolysis as illustrated in Figure 5. This would explain the large numbers of miscleaved tryptic peptides formed that bear 3−5 limit peptides. The rate at which this occurs could be slower than the rate of proteolysis in some cases. It also shows that trypsin has the option of acting as an endo- or exopeptidase in this secondary set of cleavages. Subsequent to the second cleavage, peptides are released from the larger fragment and become available for further degradation of the miscleaved peptides. It also shows that the number of tryptic peptides in the solution escalates rapidly as proteolysis progresses. The significance of this fact is that the peptide products of trypsin cleavage bind reversibly to the active site of the enzyme. This causes a rapid escalation in inhibition of the enzyme as proteolysis progresses, causing proteolysis to slow before all the tryptic peptides with missed cleavages are converted to limit peptides. The data presented here support the model that proteins are initially cleaved at a small number of favorable cleavage sites that correlate strongly with protein structure, and that reagents or conditions which modify protein conformation can alter the favorability of sites for initial cleavage. It is envisioned that basic amino acids in these sites are available for proteolysis because they are weakly associated with the protein surface, if at all. Subsequent to initial proteolysis, further hydrolysis seems to advance outward from initial cleavage sites. Again, protein structure seems to play a role, regulating the availability of peptide chains to be available for proteolysis. Native Protein Proteolysis. A central dogma in proteomics is that proteins must be reduced before trypsin digestion to allow sufficient peptides to be released for identification. This stems from the idea that protein structure is so protected by disulfide bonds they must be removed to open the structure sufficiently for trypsin to gain access to shielded lysine and arginine residues. Data from this study show that with transferrin and yeast proteins bearing multiple disulfide residues it is still possible to achieve trypsin digestion comparable to what is seen with reduced proteins. Moreover, heat based denaturation is equivalent or superior to that possible with 1 M urea. Native protein proteolysis has several attractive features. One is that different portions of the primary structure are seen compared with reduction. Second, one can use this approach to examine the disulfide structure of proteins although that was not done in these studies. The third is that it is faster. The 30−60 min of time required for reduction and alkylation is circumvented, and proteolysis can be achieved in

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CONCLUSIONS It is concluded that the degree to which a protein is unfolded at the start of proteolysis plays a role in the sequence of cleavages the molecule undergoes on the way to the final trypsin digest. Although it is not critical that a protein be reduced and alkylated before proteolysis, there is a major difference in the route of proteolysis after disulfide bond reduction. Proteolysis appears in the case of transferrin to start at a small number of preferred sites on the surface at which the polypeptide backbone is sufficiently disengaged from the surface to facilitate entry into the active site of trypsin. Proteolysis then radiates outward from these sites. It appears the structure of the protein after cleavage at favored sites controls the size of peptide fragments removed from the protein in a secondary cleavage. Reduction of disulfide bonds lead to a completely different set of favored cleavage sites. We further conclude that elevating the temperature of proteolysis increases protein digestion, more by altering protein structure and demasking cleavage sites than by enhancing enzyme reaction rate. Although the presence of disulfide bonds restricts proteolysis to some extent, the extensive disulfide bonding in transferrin did not preclude sufficient peptide generation at room temperature that the protein could be identified easily. At elevated temperature disulfide bonds still play a role in the pathway by which transferrin is digested but are far less important in determining the extent of proteolsyis. We conclude that elevated temperature is an important tool in protein proteolysis and that it will be applied increasingly in the direct analysis of native proteins. On the basis of the work reported here there is no reason reduction and alkylation must be used in protein identification.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 765-494-3878. E-mail: [email protected]. Notes

Fred E. Regnier is a cofounder of Perfinity Biosciences.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of this w o r k b y t h e N a t i o n a l Ca n c e r I n s t i t u t e ( G r a n t 1U24CA126480).

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REFERENCES

(1) Anderson, N. L.; Anderson, N. G.; Haines, L. R.; Hardie, D. B.; Olafson, R. W.; Pearson, T. W. J. Proteome Res. 2004, 3, 235−244. (2) Brownridge, P.; Beynon, R. J. Methods (Amsterdam, Neth.) 2011, 54, 351−360. (3) Reinders, J.; Lewandrowski, U.; Moebius, J.; Wagner, Y.; Sickmann, A. Proteomics 2004, 4, 3686−3703. (4) Vorob’ev, M. M. J. Mol. Catal. B: Enzym. 2009, 58, 146−152. (5) Prokai, L.; Stevens, S. M.; Rauniyar, N.; Nguyen, V. J. Proteome Res. 2009, 8, 3862−3871. (6) Rivera-Burgos, D; Regnier, F. E. Anal. Bioanal. Chem. 2012, submitted for publication. (7) Slysz, G. W.; Schriemer, D. C. Rapid Commun. Mass Spectrom. 2003, 17, 1044−1050.

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(8) Shen, Z. M.; Yang, J. T.; Feng, Y.; Wu, C. S. C. Protein Sci. 1992, 1, 1477−1484. (9) Dormady, S. J.; Lei, J.; Regnier, F. E. J. Chromatogr., A 1999, 864, 237−245. (10) Patterson, S. D.; Katta, V. Anal. Chem. 1994, 66, 3727−3732. (11) Crimmins, D. L.; Saylor, M.; Rush, J.; Thoma, R. S. Anal. Biochem. 1995, 226, 355−361.

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Native Protein Proteolysis in an Immobilized Enzyme Reactor as a

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