N-terminal sequence analysis of proteins and peptides

Abstract

Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N-terminal end. This unit describes the sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Perkin-Elmer Procise Sequencer. Sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Hewlett-Packard Model G1005A sequencer is also described. Methods are provided for optimizing separation of PTH amino acid derivatives on Perkin-Elmer instruments and for increasing the proportion of sample injected onto the PTH analyzer on older Perkin-Elmer instruments by installing a modified sample loop. The amount of data obtained from a single sequencer run is substantial, and careful interpretation of this data by an experienced scientist familiar with the current operation performance of the instrument used for this analysis is critically important. A discussion of data interpretation is therefore provided. Finally, discussion of optimization of sequencer performance as well as possible solutions to frequently encountered problems is included.

Figure 11.10.1

Edman chemistry and automated N-terminal sequence analysis. Modification of the free N-terminus of a protein or peptide sample with phenylisothiocyanate (PITC) at high pH, followed by acid cleavage of the modified terminal residue, results in release of a peptide one residue shorter in length and with a free N-terminus. This process represents a single sequencer cycle; subsequent residues can be removed by repeating this series of reactions (sequencer reactions). The cleaved 2-anilino-5-thiazolinone derivative (ATZ amino acid) is extracted from the sample support and transferred to a “flask” for conversion to the phenylthiohydantoin (PTH) amino acid (conversion reactions). The sample is then injected onto an HPLC column connected in line to the sequencer for separation analysis (PTH amino acid analysis).

Figure 11.10.2

(modified to match modified figure) Sequencer sample cartridges. (A) Glass fiber filter (GFF) sample cartridge for the Applied Biosystems Procise sequencer, used to analyze samples in solution. The sample is loaded onto a Polybrene-treated GFF, inserted into the top reaction cartridge block, and the two halves of the cartridge are sealed with a Zitex (Teflon) cartridge seal. (B) Blott cartridge for an Applied Biosystems Procise sequencer, used to analyze proteins bound to PVDF membranes, which are inserted into the semicircular slot in the top of the Blott cartridge.

Figure 11.10.3

Applied Biosystems Procise 494 reagent schematic, illustrating the current complexity of instrumentation used for automated sequence analysis. Bottles for chemistry involved in sequencer reactions include: base (R2) and PITC (R1); trifluoroacetic acid (R3) for cleavage, which can be delivered in either the gas phase or as a small pulse of liquid reagent; solvents for removing reaction byproducts (S1 and S2) and extraction of the cleaved amino acid derivative to the conversion flask (S3); and additional reagent positions for addition of optional chemistries or solvents (X1 and X3). The conversion reactions section includes: aqueous acid (R4) for conversion of the amino acid derivative to PTH amino acids; acetonitrile/water (S4) to redissolve the PTH amino acids for HPLC analysis; PTH standard (R5) used for calibration; and additional positions for user-designated chemistries or solvents (X2 and X3). Delivery of appropriate chemicals or solvents to the sample cartridges or conversion flask are regulated by the related reagent or solvent valve blocks. The HPLC injector transfers the PTH amino acids from the conversion flask to an HPLC column. The HPLC pumps and detector are controlled through the sequencer computer during the sequencing process in the automated mode. Adapted with permission from Applied Biosystems Procise User’s Manual.

Figure 11.10.4

Chromatogram of PTH standard, illustrating an example of a typical separation of PTH amino acids and Edman degradation byproducts for the Applied Biosystems Procise Models 491, 492, and 494 sequencers. The common amino acids recovered during the sequencing process are well separated, as are the normal byproducts of the Edman process: DMPTU (dimethylphenylthiourea), DPTU (diphenylthiourea), and DPU (diphenylurea). Unmodified cysteine is completely destroyed. Dithiothreitol (DTT) is typically present in the R4 reagent. S′ is an adduct between DTT and PTH-dehydroalanine, the latter of which is a degradation product of PTH-serine. Unmodified cysteine also forms S′, and can be distinguished from a serine residue because only the S′ peak increases in cycles containing unmodified cysteine. However, assigning cysteine based solely on this minor, often somewhat variable peak is risky. Abbreviations: mAU, milli-absorbance units; see Table A.1A.1 for one-letter amino acid abbreviations.

Figure 11.10.5

Effects of repetitive yield on number of cycles assigned. Theoretical amino acid yields for several different repetitive yields that are frequently encountered in automated sequence analysis illustrate the importance of this parameter on the amount of sequence that can be assigned. An initial yield of 10 pmol and a sequence detection limit of 0.5 pmol were used for this example.

Figure 11.10.6

Yields of alanine and threonine from a sequence exhibiting somewhat higher-than-normal carryover. Uncorrected values of Ala (crosses) and Thr (triangles) in each cycle of a sequence are shown. The amount of carryover in this sequence was larger than the average for this instrument, but within the range of carryover seen with some samples. The high carryover in later cycles complicates sequence interpretation. This sequence contained Ala at cycles 3, 13, and 23, and Thr at cycle 14.

Figure 11.10.7

Chromatograms of the first four cycles from two different low-pmol-level sequences. (A) 10 pmol β-lactoglobulin were loaded onto a Procise 494. The first four residues can be clearly identified above a moderate initial background. (B) A tryptic peptide loaded to the same sequencer. In this case, the high background starting in cycle 1 interferes with sequence assignment of early cycles and the first unambiguous assignment that can be made is E, in cycle 4. Abbreviations: mAU, milli-absorbance units; see Table A.1A.1 for one-letter amino acid abbreviations. All other tables unchanged from original 1997 pub.