Created: 17th December 1997, last updated: 18th January 1999,©1998 ABRF

(1) W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, CT 06510 (2) Protein/DNA Technology Center, The Rockefeller University, New York, NY 10021 (3) Department of Biology, Technion, Haifa 32000 Israel (4) Genentech Inc., South San Francisco, CA 94080 (5) Harvard University, Cambridge MA 02138 (6) Amgen Inc., Thousand Oaks, CA 92320 (7) Protein Structure Core Facility, University of Nebraska Medical Center, Omaha, Nebraska 68198-4525

Abstract

The ABRF-97SEQ sample was the 10th in a series of studies designed to aid ABRF participant laboratories in determining their abilities to obtain amino acid sequence data. The sample for 1997 was a mixture of 2 peptides at an approximate pmole ratio of 10:2, and was indicative of a peak that might be obtained by RP-HPLC of a tryptic digest. Participants were asked to use Edman sequencing or a combination of Edman sequencing and MS/MS or post source decay (PSD) sequence analysis to identify the primary amino acid sequence of the peptides. Cysteine was derivatized to Cys-S-propionamide prior to sending out the sample and a PTH-Cys-S PAM standard was included to locate the elution position of this derivative on the participant's HPLC system. An internal standard (containing norleucine and succinylated lysine) was sent as a control for the instrument's performance. This was to be co-sequenced with the unknown sample. A total of 50 responses were returned for this study, with all participants performing Edman sequencing. The accuracy of the positive correct identifications was 91.5% with 8 participating laboratories correctly identifying the major sequence. Cysteine identification improved from the ABRF-95SEQ sample which contained an unmodified cysteine, and tryptophan identification was similar to that observed in previous studies.

Introduction

ABRF-97SEQ represents the tenth in a series of unknown samples which have been distributed to members of the Association for Biomolecular Resource Facilities that perform protein sequencing. These samples are designed to allow member laboratories a mechanism with which to evaluate themselves using a sample that presents some of the problems facilities routinely encounter. Previous samples have examined the issues of sensitivity of protein sequencing (1,2), sample heterogeneity (3,4), protein-bound peptides on PVDF membrane or in solution (5,6), post-translational modifications (7), identification of cysteine and tryptophan (4,8,9), length of sequence assignment (4), and accuracy of sequence calls from a dataset (9). The current sample was designed to examine the ability of participating labs to sequence low level mixtures of peptides (a situation often encountered in core facilities) along with their ability to identify tryptophan and derivatized cysteine and be compatible with MS/MS and PSD (post source decay) techniques.

In addition, the sample was designed to be similar in composition and quantity to the peptide mixture used as dataset B in the ABRF-96SEQ sequence calling study. This allowed some comparison of sequence calling expertise with the technical aspect of sequencing peptides.

Another goal of this study was to determine the success rate of cysteine determination when a pre-derivatized sample was provided. Thus, the cysteines in the sample were modified using acrylamide to Cys-S-propionamide prior to sending out the sample. A PTH-Cys-S-PAM standard was supplied along with the sample so that participant laboratories could first determine the separation of this cysteine derivative on their sequencing systems. In addition, an internal sequencing standard was also supplied. This standard, which participants were asked to co-sequence with the ABRF-97SEQ sample, allowed an independent monitoring of the sequencer performance. Another goal of the study was to help participants correctly utilize the information in a MALDI-MS spectra of ABRF-97SEQ that was provided. Participants were asked to use this data to assist in determining the length of the peptide and to help verify that the correct sequence had been called.

Materials and Methods

A. Design and Synthesis of ABRF-97SEQ

The composition of ABRF-97SEQ was similar to ABRF-96SEQ B with the major 21-mer peptide containing two tryptophans, one early in the sequence and the other later, and two cysteines. The sequence was designed to reduce the number of lysines in the sample (for simplification of MS/MS and PSD sequencing) and for ease of synthesis. The composition of the minor 14-mer sequence was identical to the minor sequence in ABRF-96SEQ B with one arginine removed. Both peptides (Figure 1) were synthesized by Janet Crawford at the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University on a Rainin Symphony Multiple Peptide Synthesizer. Double coupling and standard fmoc chemistry was used. The protonated average mass of the 21-mer was 2509.8 amu and the 14-mer, 1502.62 amu.

Figure 1. Major and minor sequences used in the ABRF-97SEQ sample. The above two synthetic peptides were mixed at a picomolar ratio of 10:2 (major:minor) for the ABRF-97SEQ sample.

 

Crude peptides were purified on a Waters 600E system using a 20 x 250 mm YMC reversed phase HPLC column (YMC Inc., Wilmington NC) eluted at 7 ml/min. Buffer A consisted of 0.06% trifluoroacetic acid, water and buffer B, 0.052% trifluoroacetic acid, 80% acetonitrile, 20% water. A linear gradient from 0-98% B was used over 300 minutes.

B. Alkylation, HPLC Purification of Peptides, and Sample Preparation

The 21-mer peptide used as the major sequence contained two cysteines and was derivatized using acrylamide by Joe Fernandez at Rockefeller University prior to sending out the samples. The dried peptide was reconstituted in 200 mM Tris-HCl, pH 8.0 / 100 mM DTT, and reduced by incubation at 55^oC for 30 minutes. After the sample was cooled, 6 M acrylamide was added for a final acrylamide concentration of 2M, and the sample incubated at 37^oC for one hour. After alkylation, the 21-mer and 14-mer peptides were purified on a 4.6 x 250 mm Vydac C18 reversed phase column (Separations Group, Hesperia, CA). The gradient used was as follows with the buffers indicated above: 0-15 min (2-37% B), 15-125 min (37-45% B), 125-128 min (75-98% B).

Amino acid analysis was performed by standard techniques to determine the peptide concentrations. The peptides were then mixed at an approximate 10:2 ratio of the 21-mer to the 14-mer. Sequencing was performed by Edman, MS/MS and PSD by the Protein Sequence Research Committee members to ensure sample quality.

C. Sample Distribution

Samples were distributed to 215 ABRF member laboratories, which were asked to sequence the mixture either by Edman chemical sequencing, MS/MS, PSD or a combination of these techniques. In the case of Edman sequencing, 5 picomoles of an internal standard was to be added when loading the sample on the sequencer. This 17-mer internal sequencing standard contained norleucine in positions 1, 6, 11 and 16 and succinylated lysine in the remaining positions (10). Results were reported to a third party, who removed identifying marks and forwarded the data to the sequencing committee for analysis.

Results

Of the 215 facilities that were mailed the ABRF-97SEQ sample, 50 returned results. These sequence calls were scored as Positive Correct (PC); Positive Wrong (PW); Tentative Correct (TC); Tentative Wrong (TW); Overcall (OC); and no assignment. PW, TC and TW included calls of (end) before the end of the peptide. Table 1 summarizes the sequence assignments made for ABRF-97SEQ Major and Minor.

Table 1. Summary of sequence assignments for ABRF-97SEQ compared to ABRF-96SEQ dataset B. Assignments were categorized as Positive Correct (PC), Positive Wrong (PW), Tentative Correct (TC), and Tentative Wrong (TW). R is the total number of responses, which was 50 for ABRF-97SEQ and 95 for ABRF-96SEQ B. Overcalls are indicated as OC and positive/tentative assignments made in the overcalls are reflected in the total PC, PW, TC, and TW numbers.

To download the data complete with Facility identification codes, contact http://www.abrf.org/ABRF/ResearchCommittees/97psrcposter/prseq97poster.html

 

A. ABRF-97SEQ Major Sequence

The overall accuracy for ABRF-97SEQ Major was 91.8% for positive calls, with 27 out of the 50 responses containing no positive incorrect calls. There were eight responses that called the entire sequence correctly (PC and TC included), and two responses that reported no positive correct calls. One of these responses did use the internal sequencing standard, which sequenced well and indicated that the problem was not due to a sequencer malfunction, but to the sample. Thus, either the sample did not get onto the sequencer, or the eppendorf tube contained no sample. The average number of cycles called was 17 out of a total of 21 residues. The residues that were identified correctly (PC+TC) most often were alanine in cycle 8 (48/50 responses); methionine in cycle 5 (47/50); glutamic acid in cycle 6 (46/50); and glycine in cycle 7 (45/50). The residues that were incorrectly (PW+TW) identified the most were isoleucine in cycle 1 (10/50); tryptophan in cycle 16 (15/50); and serine in cycles 10 ( 10/50) and 15 (6/50). No assignment was made for 54 residues, and 11 responses (22%) had 16 residues of overcalled sequence.

Figure 2. Bar graph results of sequence calls for ABRF-97SEQ major. The correct sequence for ABRF-97SEQ major is indicated on the bottom of the graph. Sequence calls are indicated as described in the Table 1 legend, except for the "-" which is a no call.

 

B. ABRF-97SEQ Minor Sequence

For the minor sequence, there was a 74.2% accuracy of positive calls, with 12 out of 50 responses making 100% correct calls. Three responses out of the 50 called the entire minor sequence correct and 22 out of 50 responses made no positive correct calls. This lower % accuracy of positive calls is most likely due to the low amount (~ 2 pmoles) of the minor peptide. There were four respondents that did not assign any minor sequence. The valine in residue 8 was identified correct (PC+TC) most often (26/50), followed by glutamic acid in position 4 (22/50). The histidine at residue 1 was called incorrect most frequently (22 of 50 responses), and had 19 "no assignment" responses. Only one respondent identified His in cycle 1 as positive correct. Since free amino acids associated with the sample elute in the first cycle, this residue is often difficult to call. This, combined with the low amount of the minor sequence, and the low recovery of histidine when compared with other amino acids, added to the difficulty of assigning this residue. Residue 9 of the minor sequence was called incorrectly 21 times out of 50 responses. The assignment of alanine at residue 9 was complicated due to the carryover of the alanine from residue 8 in the major sequence.

There were three responses that called both the major and minor sequence entirely correct (PC+TC). Due to the low level of the minor sequence, it was not anticipated that both sequences could be called 100% correct. However, with the use of mass spectrometry, which all three labs routinely use, these respondents were able to correctly identify both of the peptide sequences.

 

Figure 3. Bar graph results of sequence calls for ABRF-97SEQ minor. The correct minor sequence for ABRF-97SEQ is indicated along the bottom of the graph. Correct sequence assignments are indicated as described in the Table 1 legend, except for the "-" which is a no call.

 

C. Comparison of ABRF-97SEQ with ABRF-96SEQB

As indicated previously, this year's sample (ABRF-97SEQ) was designed to be similar to the previous study (ABRF-96SEQ dataset B) where a sequencing dataset, instead of a sample, was sent out. The goal was to try to determine if participating facilities were having difficulties with sample handling/instrumentation or data interpretation. This was of interest since in the ABRF-95SEQ study, the positive accuracy ( PC/(PC+PW) ) was only 78%, which was about 20% less than the positive accuracy observed in previous year's studies. As indicated in Table 1, a greater number of amino acid residues were positive correctly called by participants in 1996 for both the major and minor sequences. This was due in part to having approximately half the number of responses, and one less amino acid in the ABRF-97SEQ study. The average number of correct cycles called in the major sequence was 14.5 residues in the ABRF-97SEQ study and 20.9 residues in the ABRF-96SEQ study. The minor sequence had an average number of correct calls of 4.8 residues (ABRF-97SEQ) and 10.7 residues (ABRF-96SEQB). Since the peptide compositions were similar, this suggests that participating labs are having difficulty with sample handling and/or instrument performance. The positive correct accuracy for the major sequence was slightly lower in the ABRF-97SEQ (91.5%) than in the ABRF-96SEQ (95.8%). The minor sequence also had a higher accuracy in the ABRF-96SEQB than in the ABRF-97SEQB (87.6% versus 71.7% respectively). The tentative calls were approximately equal for both studies, major and minor.

The ABRF-96SEQB dataset was obtained from a sequencing run done on an Perkin Elmer/Applied Biosystems Procise (Model 494) protein sequencer operated in the gas phase mode. The 21 responses in the ABRF-97SEQ study that were run on ABD 49X-HT sequencers (similar to that used for the ABRF-96SEQB) had about the same % positive accuracy for both the major and minor sequences (96.5% and 80.8%) as that observed in the ABRF-96SEQB study (95.8% and 87.6%). In addition, the average number of positive correct calls using ABD 49X-HT sequencers (ABRF-97SEQ) was 17.0 as compared to 20.9 for the ABRF-96SEQB study. When all types of sequencers from the ABRF-97SEQ study are included in the average number of correct calls, this number drops to 14.5.

D. Cysteine and Tryptophan Identification

To further study cysteine identification, the two cysteines in the major peptide were modified using acrylamide to Cys-S-propionamide (11). A PTH-Cys-S-PAM standard was supplied so that participants could first optimize the PTH-separation of this derivative on their systems. Many participants, however, had the PTH-Cys-S-PAM derivative eluting with either DMPTU or PTH amino acids, such as PTH-Glu. Acrylamide was chosen as the modification reagent since many proteins are currently isolated by SDS PAGE prior to submission to core facilities. These samples, whose cysteines may have been modified by acrylamide to Cys-S-propionamide, are then either sequenced directly on PVDF membranes, or digested in situ in the gel or membrane, and internally sequenced. Thus, there is the possibility of sequencing a cysteine that has been modified to Cys-S propionamide.

The positive accuracy of cysteine determination in ABRF-97SEQ Major was 88% and 97% for the two cysteines at cycles 4 (C4) and 13 (C13), respectively. This accuracy is significantly better than any previous ABRF study where a sequencable sample was distributed, as can be seen in Table 2. In the ABRF-95SEQ study, where laboratories were asked to derivatize cysteines in-house prior to sequencing, the positive accuracy was only 63%, with 35% of the respondents reducing and alkylating the sample. The ABRF-94SEQ study emphasized cysteine reduction and alkylation, supplying various protocols to use (7). In this latter study, the positive accuracy was 82% for C10 and only 59% for C20. In the ABRF-96SEQB study, a dataset was provided for sequence calling and contained carboxamidomethylated cysteine. The cysteine accuracy was 99% for both C6 and C13, which is excellent. These studies indicate that cysteine derivatization is the problem with identifying cysteine, and not identification of the cysteine derivative.

Table 2. Summary of cysteine and tryptophan identification in the past four ABRF sequencing studies. The positive accuracy for cysteine and tryptophan is listed as the amino acid (C=Cys, W=Trp) followed by the cycle # of occurrence.

 

Positive identification of tryptophan was similar in ABRF-97SEQ Major and ABRF-96SEQB Major when it appeared early in the peptide (90% and 83%). When tryptophan appeared late in the peptide, positive accuracy of identification was lower in the ABRF-97SEQ peptide sequenced in member laboratories (64% compared to 92%). This is consistent with results observed in previous studies (see Table 2) where ABRF-94SEQ W9 had an 82% positive accuracy, while at W23, only a 44% accuracy. ABRF-95SEQ contained two tryptophans late in the sequence which were again at lower accuracies of 65% (W19) and 61% (W20).

E. Sequencing Standards

The purpose of including the internal sequencing standard was to provide an independent means of determining the performance of the sequencers used in the study. This standard was used by 42 out of 50 respondents (or 84%), with 33 respondents finding it useful. In the case of one reply, where no sequence was observed, the internal sequencing standard was observed. Thus, the problem was with the sample and not with the instrument. Another response that had no positive correct calls did not make use of the internal sequencing standard and thereby, the instrument performance could not be determined. The main problem encountered with the internal sequencing standard was that norleucine was already used as a standard.

The PTH-Cys-S-PAM standard was used by 40 (80%) labs. On the majority of the systems, this derivative eluted between PTH-Glu and PTH-His for ABD systems, and a few on top of DMPTU. One lab placed it between PTH-Gly and PTH-Glu. Most Hewlett Packard users found it eluted before PTH-His.

F. ABRF-97SEQ and Mass Spectrometry

Provided with the ABRF-97SEQ sample was a matrix-assisted laser desorption/ionization (MALDI) mass spectrum of the sample. The average masses observed were 1502.1 and 2510.0 ± 1 atomic mass unit. Participants were asked to use this information to assist them in determining the approximate length of the peptides and to help verify that the correct sequence had been called. A total of 41% of the participating labs routinely use MS for estimating sample purity, peptide length or to eliminate Edman ambiguities. Seven labs (14%) routinely use MS/MS or PSD, and nine labs (18%) ran their own MS analysis of the ABRF-97SEQ sample. Six of these runs were by MALDI-MS and three used electrospray MS (ESI/MS).

There were 11 responses that overcalled the sequence. Of these 11, two responses indicated routine use of MS in their labs. Interestingly, these two sequence responses matched the predicted mass of 2510 within 1 or 2 amu, but had made errors earlier in the sequence which led to an overcall of the sequence.

ABRF-97SEQ was designed to be compatible with MS/MS and PSD analysis. Thus, it was anticipated that a few labs (particularly the seven that indicated they routinely do MS/MS or PSD) would sequence the ABRF-97SEQ using these techniques, or a combination of MS/Edman sequencing. Since only one of the 50 participating labs used MS/MS or PSD on this sample, the committee is led to believe that these techniques are not routinely used for obtaining primary sequence information.

Figure 4 contains the MS/MS spectra of ABRF-97SEQ. This spectra was obtained on 1µl of a 1 pmol/µl solution using nanospray on a Sciex triple quadrupole mass spectrometer which was operated by John Stults at Genentech. The triply charged species at 837 was fragmented. As indicated in the figure, most of the Y and B ion series ions were observed. This was sufficient data to enable the entire sequence of ABRF-97SEQ major to be determined, with a tentative assignment of Glu in position 6. Knowing the mass of the peptide, residue 6 was confirmed to be Glu. This data has been included as an example of the manner in which MS/MS can be used to obtain or confirm peptide sequence. However, it should be noted, that in many cases, the MS/MS or PSD data are not of sufficient quality to interpret the entire primary sequence. A combination of Edman and MS/MS and/or PSD data can often be used to fill in holes obtained in an Edman sequence.

Nano ESI MS/MS on a Sciex Triple Quadrupole of 837 [M+3H]3+ from the Major Peptide

 

Figure 4. Nanospray MS/MS of ABRF-97SEQ. The above MS/MS data was obtained on a Sciex triple quadropole mass spectrometer that was equipped with a nanospray source. Y and B ions observed are indicated above. Click on the image to see a higher resolution version. (Warning: the larger version of this figure is 126K and may take some time to download!)

 

Discussion

In the ABRF-97SEQ study, eight labs were able to report the entire major peptide sequence correctly, while three of these labs also assigned the minor sequence correctly. The latter three labs all used ABD 494 HT sequencers. As expected, the positive accuracy of the major 10 pmol sequence (91.8%) was higher than the minor 2 pmol sequence ( 74.2%). But, the positive accuracy of the ABRF-97SEQ was lower than that observed in the ABRF-96SEQ dataset B (95.8%). In light of the better accuracy and number of residues called in the ABRF-96SEQ B 10 pmole sample versus the ABRF-97SEQ 10 pmol sample, it appears that problems at this level are due to instrumentation and sample handling rather than data interpretation.

Cysteine derivatization appears to be the difficult step in cysteine determination since the cysteine accuracy for both an early and late cysteine in ABRF-97SEQ was quite high (C4=88% and C13=97%). Both of these cysteines were derivatized using acrylamide to Cys-S-propionamide prior to sending out the ABRF-97SEQ sample. The positive accuracy of tryptophan identification was about the same as seen in previous studies where five times more material was sequenced. Therefore, the 10 pmol amount did not appear to interfere with tryptophan identification.

Surprisingly, little use was made of mass spectrometry in analyzing this sample. Mass spectrometry is used only in a minority of protein sequencing facilities, as indicated by the survey responses. The committee feels this is a valuable tool and would expect wider use. Mass spectrometric analysis can complement Edman sequencing by providing an estimate of the number of Edman cycles needed prior to performing Edman degradation. A comparison of the observed mass of a peptide with the mass calculated from the Edman assignment can provide increased confidence of sequence assignment. Mass spectrometric analysis can often determine a non-quantitative estimate of the number of species present in the sample. Peptide sequencing by MS/MS has some advantages over Edman sequencing. MS/MS analysis often requires less material and blocked peptides can generally be sequenced. Modified amino acids and amino acids that often result in low recovery by Edman degradation (such as tryptophan, serine, threonine, and underivitized cysteine) are often easier to assign by MS/MS analysis. This technique can also yield sequence information from peptide mixtures and holes/ambiguities in Edman assignments can sometimes be determined. Sequencing by Edman degradation has some advantages over MS/MS analysis since good mass accuracy is needed to distinguish Asn/Asp and Gln/Glu. Most MS/MS methods can not distinguish amino acids such as Ile/Leu and Gln/Lys which have the same mass. The major problem of MS/MS analysis of peptides is that often not all peptide bonds are observed, resulting in a gap in the sequence. In summary, both Edman and mass spectrometry data are complementary and essential techniques in protein characterization and should be used together whenever possible.

References

1. Niece, R. L., Williams, K. R., Wadsworth, C. L., Elliott, J., Stone, K. L., McMurray, W. J., Fowler, A., Atherton, D.A., Kutney, R. and Smith, A. J. A. Synthetic Peptide For Evaluating Protein Sequencer and Amino Acid Analyzer Performance in Core Facilities: Design and Results. In (T. E. Hugli, ed.) Techniques in Protein Chemistry, Academic Press, San Diego, pp 89-101(1989).

2. Rush, J., Andrews, P. C., Crimmins, D. L., Gambee, J. E., Grant, G. A., Mische, S. M. and Speicher, D. W. A Synthetic Peptide for Evaluating Protein Sequencing Capabilities: Design of ABRF-93SEQ and Results. In (J. W. Crabb, ed.) Techniques in Protein Chemistry V, Academic Press, San Diego, pp 133-141 (1994).

3. Speicher, D. W., Grant, G. A., Niece, R. L., Blacher, R. W., Fowler A. V. and Williams, K. R. Design, Characterization and Results of ABRF-89SEQ: A Test Sample for Evaluating Protein Sequencer Performance in Protein Microchemistry Core Facilities. In (T. E. Hugli, ed.) Current Research in Protein Chemistry, Academic Press, San Diego, pp 159-166 (1990).

4. DeJongh, K. S., Fernandez, J., Gambee, J. E., Grant, G. A., Merrill, B., Stone, K. L. and Rush, J. Design and Analysis of ABRF-95SEQ, a Recombinant Protein with Sequence Heterogeneity. In (D. Marshak, ed.) Techniques in Protein Chemistry VII, Academic Press, San Diego, pp 347-358 (1996).

5. Yuksel, K. U., Grant, G. A., Mende-Muller, L. M., Niece, R. L., Williams, K. R. and Speicher, D. W. Protein Sequencing from Polyvinylidenedifluoride Membranes: Design and Characterization of a Test Sample (ABRF-90SEQ) and Evaluation of Results. In (J. J. Villafranca, ed.) Techniques in Protein Chemistry II, Academic Press, San Diego, pp 151-162 (1991).

6. Crimmins, D. L., Grant, G. A., Mende-Muller, L. M., Niece, R. L., Slaughter, C., Speicher, D. W. and Yuksel, K. U. Evaluation of Protein Sequencing Core Facilities: Design, Characterization, and Results form a Test Sample (ABRF-91SEQ). In (R. H. Angeletti, ed.) Techniques in Protein Chemistry III, Academic Press, San Diego, pp 35-35 (1992).

7. Mische, S. M., Yuksel, K. U., Mende-Muller, L. M., Matsudaira, P., Crimmins, D. L. and Andrews, P. C. Protein Sequencing of Post-translationally Modified Peptides and Proteins: Design, Characterization and Results of ABRF-92SEQ. In (R. H. Angeletti, ed.) Techniques in Protein Chemistry IV, Academic Press, San Diego, pp 453-461 (1993).

8. Gambee, J. E., Andrews, P. C., Grant, G. A., Merrill, B., Mische, S. M. and Rush, J. Assignment of Cysteine and Tryptophan Residues during Protein Sequencing: Results of ABRF-94SEQ. In (J. W. Crabb, ed.) Techniques in Protein Chemistry VI, Academic Press, San Diego, pp 209-217 (1995).

9. Fernandez, J., Admon, A., DeJongh, K., Grant, G., Henzel, W., Lane, W. S., Stone, K. L. and Merrill, B. Evaluation of ABRF-96SEQ: A Sequence Assignment Exercise. In (D. R. Marshak, ed.) Techniques in Protein Chemistry VIII, Academic Press, San Diego, pp 69-78 (1997).

10. Elliott, J. I., Stone, K .L. and Williams, K. R. Synthesis and Use of an Internal Amino Acid Sequencing Standard Peptide. Anal. Biochem. 211: 94-101(1993).

11. Brune, D. C. Alkylation of Cysteine with Acrylamide for Protein Sequence Analysis. Anal. Biochem. 207: 109-116(1992).

David W. Speicher

Wistar Institute Philadelphia, PA, USA

speicher@wista.wistar.upenn.edu