Ken Williams1, Ryuji Kobayashi2, William Lane3, and Paul Tempst4
1W.M. Keck Biotech. Resource Lab. at Yale U.
2Cold Spring Harbor Laboratory
3Harvard Microchemistry Facility
4Memorial Sloan Kettering Cancer Center
Since many eukaryotic proteins have blocked NH2-termini (1, 2), there is considerable interest in developing better procedures for isolating internal peptides suitable for amino acid sequencing. The capabilities of micro-sequencing are such that "preparative" amounts of protein can now be isolated by SDS PAGE and, indeed, the latter appears to be the current method of choice for bringing about the final purification of low abundance proteins. Hence, procedures for generating internal peptides from proteins that have been separated by SDS PAGE are generally the most useful. As described in last year's ABRF Workshop entitled "Digestion of Proteins from Blots and Gels" (3), several approaches may be taken to obtain peptides from SDS PAGE separated proteins. These include, but are not limited to: 1) digest the protein in the gel in the presence (4) or absence of SDS (5, 6) and then diffuse the resulting peptides out of the gel; 2) blot the protein onto nitrocellulose (7, 8) or PVDF (8) and then digest it in situ and, 3) blot the protein onto PVDF, cleave it in situ with cyanogen bromide, elute the resulting peptides and then further digest them with trypsin (9). Each of these procedures has its own advantages and disadvantages with the impetus towards deriving better "in gel" techniques deriving from the observation that electroblotting is often not quantitative. Ultimately, the rate of proteolysis and thus the success of any of these procedures depends upon maintaining the highest possible concentration of susceptible peptide bonds and of the protease that is being used. Hence, using a 1:25 (w:w) ratio of trypsin in 2 M urea, the substrate concentration should be kept above 20 ug/ml (10). Obviously, every effort should be made to keep both the substrate and enzyme concentration as high as possible. However, methodological constraints often limit the substrate concentration and the enzyme concentration is limited somewhat by the generation of autolysis products. Other important considerations are that the protein be denatured, yet soluble, and that either the SDS is removed prior to digestion or that an enzyme such as lysylendopeptidase is used that is active in the presence of SDS. In the latter case, the SDS is removed by means of an anion exchange precolumn (4). This article provides an overview of the internal amino acid sequencing techniques that are being used in four laboratories and of the results that are being achieved. Our purpose is to 1) demonstrate that several different approaches can be used to carry out high sensitivity internal sequencing on a wide range of "unknown" proteins, 2) share a few observations that laboratories using these procedures have made regarding their optimization and finally, 3) to provide at least semi-quantitative data regarding the limits of these approaches.
The Microchemistry Facility at Harvard (Bill Lane, Director; Renee Robinson; Mary Gordy and Violaine Bailey) uses the Fernandez et al. (8) approach (with minor modifications), predominantly with nitrocellulose (more recently, PVDF). We note that the addition of the hydrogenated Triton X-100, as recommended by Fernandez et al. (8), appears to give a 30-40% improvement in peptide yield as judged by the increase in peak height and the number of peaks recovered by RP-HPLC. Historically speaking, our advice to investigators is based on our first five years of in situ digestions on nitrocellulose, (ca. 720 samples), prior to the introduction of the Fernandez buffer (8). Our facility is, however, gradually shifting to PVDF based on the increasing evidence that peptide recovery (with the Fernandez buffer) is higher from PVDF than from nitrocellulose supports (reference 8 and D. Speicher, personal communication). In addition, PVDF allows quantitation of protein amounts via pre-digest amino acid analysis as opposed to the post-digest analyses (see below) that we currently carry out with nitrocellulose-bound proteins. However, we continue to adhere to precautions stemming from our experience with nitrocellulose to leverage any possible advantage for extremely low level samples.
The minimum exposure necessary to accomplish the task of Ponceau S staining, destaining, and washing is recommended followed by a 30-90 sec wash in 1% acetic acid. The recommended NaOH wash (7) has been deleted (after discussion with R. Aebersold) and the investigator is advised to excise the band as judiciously as possible (to limit the surface area of nitrocellulose (NC)) prior to exposing it to water. On nitrocellulose we and P. Tempst's lab (see below) have correlated greater membrane surface area with poorer peptide recovery. We note that Stone et al. (9) have previously observed a similar finding using their CnBr elution procedure from PVDF membranes. As a result of these observations, we urge investigators to overload the gel up to the extent resolution is retained and to judiciously excise any unstained or partially stained perimeter of the band as even a 0.5 mm excess perimeter on a 1 x 10 mm band doubles the surface area. After excision, the NC is placed in a 1.5 ml eppendorf tube for a brief vortex with water. Excess water is removed and the MOIST NC membrane is stored frozen to trap it in that state for shipment to the facility.
Since protein blotting is in the hands of the submitting investigator, data regarding the amount of protein subjected to SDS PAGE and blotting efficiencies are not available. We therefore take a conservative stance, strongly recommending an absolute minimum of 100 pmol of BLOTTED protein. Our suspicion, albeit speculative, and based on observation of the stained blot as submitted as well as on post-digest amino acid analysis (see below), is that on average users provide significantly less protein (i.e., 2-3 fold) than they estimate. An aliquot of the post-digest supernatant is routinely hydrolyzed and analyzed prior to RP-HPLC. Although a 1990 ABRF study (11) demonstrated that salts cause increased error in PTC amino acid analysis, this will be a consistent error which should not significantly interfere with the ability of these analyses to serve as a predictor of the relative chance of success of the impending HPLC separation. In general, the procedure appears to have an 80-90% success rate in the 40-60 pmol range with a nearly 100% success rate when the amount of protein digested is above the 100 pmol amount. The Fernandez et al. (8) procedure, which we have slightly modified, has succeeded with samples whose PTC analysis results were as low as 7-15 picomoles. As we are always conservative, if such low amounts of protein are detected we advise the investigator that it would probably be in their interest to purify additional protein prior to proceeding, thus avoiding the needless expenditure of an HPLC separation that has a low probability of success. While we have not stringently quantitated criteria for judging the success of our internal sequencing strategy with any given protein, we qualitatively judge it to be a success when we are able to provide the submitting investigator with two or more peptide sequences of sufficient length to design oligonucleotide probes, identify the protein via database searches or to synthesize peptides for the generation of antibodies.
Trypsin and lysylendopeptidase have been the most reliable enzymes in our hands. Peptides are collected on an HP1090 using automated peak detection (ISCO/Retriever II) on Vydac C18 2.1 x 150 mm columns using a flow rate of 150 ul/min so that peak volumes are 75-150 ul. Individual peaks are critically reviewed "full screen" on the data acquisition computer to better visualize details of their symmetry and resolution, particularly across multiple wavelengths obtained with diode array detection. Of the optimal fractions from this screening, 0.5 - 3 ul is taken for laser desorption mass spectrometry (LDMS) on a Finnigan Lasermat. The latter is critical for identifying peptide mixtures, maximizing sequence length and for verifying the resulting protein sequences. This amount of peptide (20 - 200 fmole based on amino acid sequencing yields) is ample for routine analysis. All of the ABI 477 sequencers use a microcartridge and fast cycles, in addition to the modifications advised by P. Tempst (12).
Finally, we stress the often uncited importance of thorough discussions with the investigator (and particularly the preparer, who is frequently someone else). Such dialogues are crucial to uncovering potential problems with their protein and preparation and allow for the best strategies for digestion and successful sequencing.
Cold Spring Harbor Laboratory uses the Kawasaki et al. (4) "in gel" approach with minor modifications (no smashing of the gel). The digest is carried out at 30deg.C for 24 h, with the resulting peptides being separated on a Vydac C-18, 300 Å, 5 micron (2.1 x 250 mm) column that is preceded by a Brownlee anion exchange cartridge column, 3.2 x 15 mm). The column is equilibrated at a flow rate of 0.2 ml/min with 2% acetonitrile, 0.1% TFA and the peptides are eluted with increasing concentrations of a 3:1 (v:v) acetonitrile/2-propanol mixture containing 0.09% TFA (alternatively, 2% acetonitrile, 0.08% TFA is used with 70% acetonitrile containing 0.073% TFA). Based on comparative in solution/in gel digests of 5 ug (~75 pmol) BSA, the overall relative peptide recovery appears to be about 80-90% (as determined by the peak height and number of peaks present in the HPLC profile). However, when 1-2 ug BSA was digested, the recoveries were less than expected based on the 5 ug sample and the yields were poor. The recovery of peptides in the extract from the gel after digestion of ~5 ug amounts of three 32[P]-labelled proteins ranged from 96-97%. We have used this approach on 39 different "unknown" proteins (that range from 14-300 kD in size) and it has succeeded in each instance. cDNA clones, which confirmed our internal sequences, have been isolated for 16 of these proteins. In addition, 6 of these proteins are now being cloned and the remaining 17 proteins were identified following database searches of our internal sequences. In general, 2-3 ug of a 50 kD protein, that is, about 50 pmol is recommended for this procedure. This method may be particularly advantageous to methods that rely on blotting when multi-protein complexes (i.e. origin recognition complexes, replication factors, chromatin assembly factors, and transcription factors) are being studied. That is, with the Kawasaki et al. (4) approach, one does not have to worry about possible differences in blotting efficiencies for different size proteins. The amount of protein is based on Lowry/Bradford assay or on relative Coomassie Brilliant Blue G staining intensity, both of which often show substantial error when compared with amino acid analysis. Nonetheless, this procedure has succeeded with as little as 20 pmol estimated protein, however, the sequencing data is not as strong as at higher levels where it is also possible to call longer sequences. In general, priority is given to sequencing relatively long peptides so that PCR can be done within the same peptide sequence. This makes PCR cloning more straightforward since the products are of predicted length. In this respect, Achromobacter protease I (Lysylendopeptidase, obtained from Wako) offers some advantage over trypsin. The anion exchange precolumn (which removes SDS just prior to reverse phase HPLC) is somewhat problematical and needs to be replaced after five runs (or after a total of 1 mg SDS has been injected). The use of a 0.05% solution of Coomassie Brilliant Blue G (Aldrich #23,464-8) is recommended for staining as this chromatographically purified stain yields only a single, very late eluting peak following reversed phase HPLC (Hewlett Packard 1090) on a Vydac C-18 column. Peptide sequencing is carried out on an Applied Biosystems 470 sequencer with on-line PTH amino acid analysis using the manufacturer's operating program. We have recently carried out one preliminary study (using 2 ug or ~30 pmol BSA) where the Rosenfeld et al. (6) in gel procedure was compared to the Kawasaki et al. (4) in gel procedure. To better compare these two methods, we replaced the 1 hr water wash of the gel (after destaining) with two 50% acetonitrile washes (each wash was carried out for 20 min) and we removed the anion exchange precolumn prior to starting the acetonitrile gradient. Based on this limited comparison and the resulting HPLC absorbance profiles, the Rosenfeld et al. (6) procedure gave better recovery of later eluting peptides. As judged by the resulting profiles, the Rosenfeld procedure gave similar results when both 50% acetonitrile and 50% methanol were used for washing the gel prior to the digestion and no difference in recovery was noted when the Tween 20 concentration was reduced from 0.02% to 0.01%. Using a slightly modified Rosenfeld et al. procedure [i.e., 50% methanol for washing the gel, 0.01% Tween 20, lysylendopeptidase (300 ng/digestion) and 0.05% Coomassie Brilliant Blue-G (>90% pure) in 0.5% acetic acid, 20% methanol], we have now obtained satisfactory peptide maps using an estimated 1-2 ug of nine unknown proteins and three known proteins that range in size from 28 to 120 kD.
The Sloan-Kettering Microchemistry Core Facility (Paul Tempst, Director; Scott Geromanos, Manager) use the Aebersold et al. (7) approach with nitrocellulose (as described in detail in (13)). The protocol is essentially unmodified from the original procedure (7), except that a different HPLC set-up is used. Investigators are advised to electroblot in "wet" (not semi-dry) tanks at low voltage (5 hr to overnight), to stain with Ponceau S (not Coomassie Blue) and to not let the NC dry out. In agreement with Aebersold et al. (7), we have qualitatively correlated poorer peptide recoveries with letting the NC membrane dry out, hence, all subsequent NC manipulations are done while the NC is submerged in water. In those few instances where failures have occurred, invariably one of the latter recommendations has not been followed. No more than three blotted lanes are accepted with a total surface area of less than 100 mm2; a similar sized blank strip of NC, from the same blot, is also requested for a digest blank. We recommend loading a dilution series of Bio-Rad MW markers (2, 1, 0.5, 0.25 ug) in the unused lanes, leaving one blank lane on either side of the sample; wells of these blank lanes are filled with neat Laemmli buffer. We have not yet tried this approach with PVDF. Since problems have been encountered with in situ alkylations on NC (14), this procedure is carried out following digestion. The excess reducing and alkylating reagents are then removed by isocratic elution of the reverse phase HPLC column prior to starting the gradient. This approach has been used on a total of over 200 unknown proteins with a nearly 100% success rate with amounts of blotted protein that are estimated to be routinely below 25 pmol. Several digests have been carried out with less than this amount, with the most sensitive carried out on what was estimated to be ~10 pmol protein on the NC blot. Although quantitation of the amount of blotted protein is based on relative staining intensity with Ponceau S (and is therefore an approximation), this procedure has been demonstrated to succeed (as judged by the resulting HPLC profile) when 20 pmol carbonic anhydrase (29 kD) was actually loaded onto the gel. Since our facility does not offer electroblotting as a service, accurate data regarding the amounts of unknown proteins that were actually applied to the SDS PAGE gels are not available. Preliminary estimates on peptide quantities that are to be expected usually come from silver-stained pilot gels. However, based on densitometric scanning of 250 ng amounts of 10 "standard" proteins subjected to SDS PAGE in the Keck Facility there is a several fold range in the relative silver staining intensity of proteins. Peptides are collected manually from modular HPLC systems (140B syringe pump and 1000S diode array detector, both from ABI) that are equipped with 2 mm ID columns (Vydac C-4 or C-18, 25 cm length) and that are operated at a flow rate of 100 ul/min. All HPLC systems are outfitted with glass capillary outlet tubes that are plumbed directly into the flow cell, which reduces post-flow cell volumes to less than 2 ul (details on this HPLC configuration will be forthcoming in a paper by Elicone et al.). After storage, column fractions (typically 30-50 ul) are supplemented with neat TFA (to give a final concentration of 20%) before loading onto the sequencer disc, as we have shown that this often increases recoveries of low pmol amounts of peptides from 50 to 85% (14). Initial sequencing yields are usually in the 1-3 pmol range and hence, all sequencers (Applied Biosystems 477's) have been modified as previously described (12, 14). If judged necessary (and deemed feasible), peptide fractions are rechromatographed on 1 mm ID columns (operated at 30 ul/min flow rates, with an average fraction size of 15-20 ul) before sequencing.
The W.M. Keck Facility (Ken Williams, Director; Kathy Stone, Manager of Protein Chemistry) utilizes an in gel procedure (5) derived from Ward et al. (15). Like the Rosenfeld et al. (6) procedure, the W.M. Keck procedure relies on removing the SDS from the Coomassie Blue stained gel prior to in situ trypsin digestion. In the case of the Keck procedure, the protein is also reduced and carboxymethylated in situ immediately prior to digestion. After receipt of an in gel sample, ~15% of the sample is subjected to hydrolysis and ion exchange amino acid analysis to estimate the amount of protein. Although six amino acids (Gly, Met, His, Trp, Arg and Cys) usually cannot be quantitated due to their low abundance in the 0.3 ug - 2 ug aliquots of protein that are typically being hydrolyzed and/or to interference by components of the SDS gel, these analyses nonetheless provide significantly more accurate estimates of the amount of protein that is about to be digested then can be obtained by judging relative staining intensities. Hence, based on hydrolyzing and analyzing ~15% aliquots of SDS PAGE bands containing increasing amounts of transferrin, the absolute recovery of those 14 amino acids that can be readily quantitated following in gel hydrolysis (after correcting for losses based on an internal standard (norleucine) and for "background", based on analyzing an equal size, blank section of gel) increased from 75% to 95% as the TOTAL sample load increased from 4.6 to 18.4 ug. The average compositional errors for these 14 amino acids were +/-9.9% when 0.3 ug of the former sample was analyzed and +/-6.7% when 0.92 ug of the latter sample was analyzed. Based on the composition of an average eukaryotic protein, not accounting for the 6 indicated amino acids will, in general, underestimate the total amount of protein by 22%. To put this in perspective, if we were to analyze 15% of a 100 pmol sample of a 50 kD protein that was destined for in gel digestion, we would expect to recover ~80% of the protein and would be able to quantitate 78% (by weight) of the released amino acids. Hence, we would underestimate the amount of protein by 38% [i.e. 1-(0.8 x 0.78) x 100]. Obviously, by routinely adding a 22% correction for those 6 amino acids that are difficult to quantitate following in gel hydrolysis, the estimate of the total amount of protein subjected to in gel digestion would most likely be within 25% of the correct value. This compares with the several fold range we found for the relative silver staining intensity of 10 standard proteins (see above). Regardless of the absolute accuracy of the protein estimates obtained by in gel hydrolysis/amino acid analysis, they provide an extremely valuable predictor of the likely success rate of the digest and, when this analysis indicates too little protein, it provides the investigator with an opportunity to "hold" their sample until it can be pooled with additional material. The value of these analyses is further indicated by the fact that in every instance where amino acid analysis has indicated that an insignificant amount of protein was present (e.g., less than the 0.09 +/-0.07 ug of protein that the Keck Facility has found in 25 "blank" sections of gels submitted by investigators), but the investigator has insisted that the sample be digested nonetheless, the digest has failed. Along with each sample that is digested, the Keck Facility also digests a positive control (a similar number of pmol of an in gel transferrin sample prepared in the Keck Facility) and an equal volume section of "blank" gel, which is supplied by the investigator submitting the sample. The latter is useful for identifying artifact peaks due to gel components, excess reduction and carboxamidomethylation reagents, Coomassie Blue and trypsin autolysis products.
Following digestion, the peptides are recovered by diffusion prior to reverse phase HPLC on narrow bore columns (2.1 mm x 25 cm, Vydac C-18) eluted at 150 ul/min. Peptides are collected (with peak detection) into eppendorf tubes (16). Providing they are capped, these fractions may often be stored for extended periods of time (ie., 2 or more years) at 5deg. C prior to sequencing. Typically, 3 ul aliquots of 5*10 of the most symmetrical, later eluting absorbance peaks are subjected to laser desorption mass spectrometry on a Fisons TofSpec Instrument. This technique has provided usable mass data on as little as 6 fmol of a 3,000 dalton peptide (personal communication from John Rush; the signal/noise ratio was ~7 and the mass accuracy (without an internal standard) was + 0.4%) and has proven invaluable for identifying peptide mixtures and artifact peaks prior to sequencing and for confirming the resulting sequence as determined by Edman degradation. After selecting a suitable peptide based on the LDMS data, it is loaded onto an Applied Biosystems 470 or 477 sequencer. The eppendorf tube that contained the sample is then rinsed with 30 ul neat TFA and this wash is also applied on top of the sample.
The Keck Facility recommends that a minimum of 100 pmol protein be used for internal sequencing. At this level, the success rate approaches 100%. As indicated in Table I, the overall success rate in the <100 pmol range is about 75%. No data is available on the ability of the Keck Facility's in gel approach to work with less than 35 pmol amounts of "unknown" proteins. It is, however, encouraging that, as shown in Table I, there does not seem to be any significant downward trend with respect to the initial yield of the resulting peptides as the amount of protein is decreased from the 300-400 to the <100 pmol range. This suggests the limits of this approach have not yet been reached and, in fact, when 25 pmol transferrin was subjected to this procedure it worked very well based on the resulting HPLC profile. As expected, recent data suggests the density of the protein band in the gel is important. Hence, the success rate for 45-94 pmol amounts (mean = 69 pmol) of 9 unknown proteins that were submitted at densities above 0.05 ug/mm3 was 90%. In comparison, the success rate for 23-85 pmol amounts (mean = 65 pmol) of 10 unknown proteins that were submitted at densities below 0.05 ug/mm3 was only 70%. In general, investigators are encouraged to submit samples with a density of at least 0.1 ug/mm3. Additional studies are needed to determine the absolute sensitivity limits for this in gel procedure, which could be further improved by the use of thin gels as nearly all experiments carried out to date have utilized 0.8 - 1.5 mm thick gels.
While our experience suggests it is now possible for a laboratory that specializes in internal sequence analysis to usually succeed with 50 pmol amounts of protein being applied to the SDS polyacrylamide gel (provided the protein/gel or protein/membrane ratio is sufficiently high), there is nonetheless agreement that the quality of the resulting data is improved by going to the 100 pmol level. Unfortunately, neither the data in the literature nor in this article (particularly with respect to the amount of protein that was subjected to SDS PAGE) is sufficiently quantitative to be able to make a firm determination as to which of the various procedures that have been discussed provides the highest yield of peptides from a given amount of protein. The problems in this regard have already been noted in this article and are further evident in the summary in Table II. Hence, the initial protein amounts in the Fernandez et al. (8) reference are based on estimates (which are presumably derived from relative staining intensities) obtained from the investigators submitting the samples. In the case of Kawasaki et al. (4), no data is provided on average peptide sequencing yields. In the case of the Aebersold et al. (7) and Rosenfeld et al. (6) references, initial protein amounts (which are not documented with regards to how they were determined) and average peptide sequencing yields are given for only two proteins. In this article we have provided reasonably accurate estimates (based on amino acid analysis) of the overall amounts of protein that were digested by the Fernandez et al. (8) membrane approach and by the Stone et al. (9) in gel procedure. However, particularly in the case of the Fernandez et al. (8) approach, these are not the amounts of protein that were actually subjected to SDS PAGE. Indeed, when the Keck Facility submitted 15 ug amounts of 5 standard proteins (transferrin, ovalbumin, carbonic anhydrase, trypsin inhibitor and a-lactalbumin) to SDS PAGE on a 12.5% gel followed by blotting onto ProBlott, the average overall recovery was ~50% based on hydrolysis and amino acid analysis of the Coomassie Blue stained membrane. This recovery is lower than might have been expected based on the 83% blotting efficiency that has previously been reported for ProBlott (17). The differences between these two studies may result from the fact that different proteins were used or from the fact that in the case of the Keck Facility, the yield of blotted protein is being compared to the amount of protein that was applied to the gel whereas in the case of the previous report (17), it was being compared to the amount of protein in an identical gel piece that was not blotted. Keeping all these caveats in mind, the relatively narrow range of peptide recoveries (again, the latter are relative to the amount of protein digested as opposed to the amount that was actually subjected to SDS PAGE), that are listed in Table II suggests there is probably less than a 4-fold range in the average peptide recoveries obtained from a given amount of protein subjected to SDS PAGE using these various approaches. Based on the RELATIVE recoveries of peptides from in gel or on membrane approaches versus in solution digests, there may be a slight advantage in the case of the in gel approaches. That is, the relative recovery of peptides from the Kawasaki et al. (4) and Rosenfeld et al. (6) in gel procedures appears to be 80-97% (see above as well as Fig. 4 in reference 4) and 65% (6) respectively of that obtained when the same amount of protein is digested in solution without subjecting it to SDS PAGE. In contrast, the relative recovery of peptides with the Fernandez et al. (8) membrane procedure has been reported to be only 35% (from ProBlott) and with the Aebersold et al. (7) membrane procedure to be only 27% (based, however, on only 3 tryptic peptides from BSA) of that obtained from a corresponding digest carried out in solution (18). Clearly, additional studies are needed to determine if any of these procedures can routinely provide an increased yield of peptides from a given amount of protein subjected to SDS PAGE. Such a determination would probably require that parallel studies be carried out in the same laboratories on a wide variety of proteins.
One final note with regards to the recovery of peptides from proteolytic digests is that even under "ideal conditions" (ie., with 15 nmol of an in solution tryptic digest of a 36,000 dalton protein that digests well), the absolute recovery of an "average" tryptic peptide was only 52% (9) based on amino acid analysis of the resulting, HPLC purified peptides. In comparison, an average sequencing yield of 13% was obtained for tryptic peptides isolated from 67 different proteins subjected to the Stone et al. (5) in gel procedure (Table I). Assuming an average first cycle coupling efficiency of 50-75%, the actual recovery of an average tryptic peptide from this in gel approach was thus approximately 25%. Hence, regardless of how much additional research is carried out to improve this particular approach, it is unlikely that the absolute yield of an average tryptic peptide can be improved by more than two-fold over the existing procedure (5). Unfortunately, and as noted previously, absolute recoveries of large numbers of peptides (determined by the first/second cycle amino acid sequencing yields as compared to the amount of protein (estimated by amino acid analysis) subjected to SDS PAGE) are not yet available for most internal digestion protocols.
Finally, we would be remiss to not point out the enormous contribution that LDMS can make to internal sequencing. The sensitivity of this technique is such that masses can be routinely determined on 20-500 fmol amounts of most tryptic peptides (unpublished observations of the Keck and Harvard Microchemistry Facilities). The latter means that a few microliters of an HPLC fraction containing a peptide isolated from a 50 pmol tryptic digest is sufficient for a mass determination. In addition to rapidly confirming sequences determined by Edman degradation, routine "screening" of small aliquots of all peptides destined for sequencing has already proven to be extremely beneficial in terms of detecting peptide mixtures and artifact peaks prior to amino acid sequencing. (Copies of this article, including copies of the above protocols, may be obtained from Ken Williams, whose address is on page 2.)
The authors especially thank Dr. Nancy Maizels (Yale University), whose inquiries led to the writing of this ms.
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