Product
Review
Automated Protein
Sequencers Cycle On Pehr Edman's derivatization and cleavage method of sequencing proteins from the amino (N-) terminus has been around since 1950 and is still hardy. Despite ongoing problems with uneven amino acid reactivity and the detectability of the derivatives, all automated N-terminal protein sequencers still use this chemistry, with a few modifications. Today's sequencers deliver more options and offer significant improvements in performance over the previous generations of instruments. What has changed most over the years is the ability of newer systems to accommodate a wider variety of peptide purification methods, the incorporation of advanced software and hardware, and the development of sequencing techniques for proteins containing post-translational modifications and recalcitrant amino acids. New strategies have in some cases reduced the extent of sequencing performed on each sample. Instead of trying to sequence an entire protein by Edman degradation, many researchers now sequence only a few peptidefragmentsin order to synthesize the corresponding oligonucleotide probes, which they use to clone the gene for the target protein. The DNA is much simpler to sequence, and the full protein sequence can be derived from the DNA sequence.
Edman-chemistry protein sequencers continue to improve as they adapt to researchers' changing needs However, such strategies have not eliminated the demand for protein sequencing. When short sequences are needed, or when the functional protein contains changes from the putative DNAderived sequence, protein sequencing is a valuable complementary technique despite its limitations. The increased demand for peptide and protein synthesis in pharmaceutical applications and basic research has made automated protein sequencing indispensable for quality control. We asked Robert B. Harris of Commonwealth Biotechnologies (Richmond, VA) and Ronald Niece of the University of Wisconsin to discuss the current state of automated protein sequencing technology and new developments. Table 1, al-
though not comprehensive, lists a representative selection of currently available commercial systems. Sample preparation
Sample proteins are often sequenced without further treatment. However, sometimes the sample protein must be denatured. Intact disulfide bonds must be broken by reducing them and the cysteine residues alkylated to prevent degradation. In other instances, the N-terminus of the native protein contains carbohydrates or other moieties that prevent sequencing at that site, so the protein must be chemically or enzymatically cleaved into peptides to provide new starting points for sequencing. The purified peptide or protein is immobilized on a support and placed in a sequencer cartridge. Earlier systems such as the discontinued Milligen sequencer called for covalent binding on glass fiber filter disks, says Niece, but nowadays most protocols rely on hydrophobic or charge interactions to immobilize the peptide. Often the glass fiber filters are coated with polybrene, a polymer that improves retention of peptides. Newer sequencing systems accommodate various other sample supports such as polyvinylidine difluoride (PVDF) membranes for electroblots
Analytical Chemistry, Vol. 66, No. 24, December 15, 1994 1239 A
Product
Review
from gels, column packings that adsorb or covalently bind the peptides, and synthe sis resins with partially or fully synthe sized peptides still attached. Sequencing
The Edman method involves three chemi cal steps, all of which are performed in
an inert gas environment (N2 or Ar). First, the amino acid at the N-terminus of the immobilized peptide is derivatized with phenylisothiocyanate (PITC) and a base such as trimethylamine (TMA) in the derivatization cartridge. Second, the anilinothiazolinone (ATZ) amino acid deriva tive is cleaved from the peptide with tri-
fluoroacetic acid (TFA), extracted into an organic solvent, and sent to the conver sion cartridge of the sequencer. There the ATZ derivative reacts with a second dose of TFA in aqueous solution to form the more stable phenylthiohydantoin (PTH) derivative. This three-step process is repeated for each subsequent amino
Table 1 . S u m m a r y of r e p r e s e n t a t i v e products.
Product Company
LF3000 Beckman Instruments 2500 Harbor Blvd. Fullerton, CA 92634 800-742-2345
HPCrf«MA Hewlett Packard 1601 California Ave. Palo Alto, CA 94304 415-857-6100
HPQ1O0M Hewlett Packard 1601 California Ave. Palo Alto. CA 94304 415-857-6100
Price Dimensions ;w X d X h: cm) Sequencing method Sample load No. samples in queue Sample supports
$103,80O~$145.800 166X56X89
$110,000-$t25.000 275 X 77 X 77
$150.000 275 X 77 X 77
N-terminal 1 pmol-5 nmol 1 or 3 Glass fiber disks, PVDF membranes, glass beads, synthesis resins
N-terminal < 5 pmol-20 nmol 2 or 4 Biphasic hydrophobic/ hydrophilic packed column, PVDF membranes, synthesis resins On-column sample concen tration, washing, cysteine reduction/alkylation, CNBr or enzymatic cleavage Liquid-phase, programmable bidirectional fluid flow
C-terminal 1 nmol 1,2, or 4 Glass fiber disks, PVDF membranes, synthesis resins
On-column sample concen tration and washing
Oiisopropytetbylamine as base, octylamine scavenger for modified PITC added to reduce side reactions 40
Sequencing chemistry goes through proline
Concentration and desalting in a sample prep cartridge, optional covalent attachment to membrane Gas-phase or pulsed-liquid, metered fluid delivery with optical sensor monitoring N-methylptperidine base
Precycling/sample prep
No precycling required with proprietary supports that don't contain polybrene
Reagent delivery
Gas-phase, upward fluid flow, programmable
Chemistry modifications Dilute PITC. diisopropylethylamine or triethylamine as base Cycle time (min) Repetitive yield Separation system
Detector
Control system
Reagent kits Special features
Single cartridge-42; triple cartridge-47 > 94% with 10 pmol 97% with 10 pmol human (x-lactalbumin serum albumin System Gold liquid chroHP 1090M liquid chromato graph matograph, also supports Applied Biosystems t20A liquid chromatograph Variable-wavelength UV or 269-nm filter photometric dual-wavelength diode array detector detector Manual or automated control Manual or automated con of sequencer and HPLC, trol of sequencer and HPLC, 486-based PC, protocols 486-based PC with that can be changed in real Windows-based interface, time, sequence interpreta automatic interrupt function tion software $1390-$t490/kit, -750 cycles CE, second HPLC, or amino acid analyzer unit can be added; automatic N 2 tank switching
70
-30
NA HP 1090M liquid chromato graph
> 94% with 10 pmol β-lactoglobulin Model 190 microbore dualsyringe HPLC system
269-nm filter photometric detector
Variable-wavelength UV detector
Manual or automated con trol of sequencer and HPLC, 486-based PC with Windows-based interface, automatic interrupt function
Macintosh Quadra interface, preprogrammed protocols that can be changed in real time, automated sequence interpretation software, on screen temperature control, pressure and event log $732/250 cycles, $1299/500 cycles, $1657/750 cycles Reagent delivery mode switching between samples within a run, electronic pres sure management system
$2120/kit, > 700 cycles
$1680/k.it, 360 cycles
Sample prep station module, automatic Ar tank switching
Automatic Ar tank switching
NA = Not applicable
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Liquid-phase, programmable bidirectional fluid flow
Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
Precise Perkin Elmer Applied BJosystems Div. 850 Lincoln Centre Dr. Foster City. CA 94404 800-345-5224 $107,000-$135.000 138X53X61
acid in the peptide chain. The PTH amino acids from each cycle of the process are flushed onto an HPLC column for separa tion and UV detection; their retention times are used to identify them for se quence assignment.
and extra reagent bottles are usually avail able for additives and modifiers.
Yield The basic Edman method has several limi tations. Sensitivity is one; the protein pu rity required for an accurate peptide se quence often comes at the price of low Reagent delivery abundance, and obtaining sufficient puri "Basically," says Harris, "Edman chemis try is very amenable to automation. There fied protein for sequencing can be diffi cult. Reaction efficiency is another factor are lots of variations, to be sure—reagent delivery, drying solvents, extraction times, because not all amino acids are equally amenable to derivatization and cleavage and so forth—but basically each instru from the peptide chain. Those that are left ment comes with recommended pro behind in one cycle can serve to contam grams to cover a variety of experimental inate subsequent cycles, making the se situations." The choice of sample sup quence assignment less certain as the port and reagent delivery system has process continues. evolved considerably in the past 30 years—generally in the direction of reduc "Repetitive yield" determines how ing sample and reagent volumes to in many cycles can be run and how much of crease the reaction efficiency. a peptide can be sequenced. A repetitive yield below 90% will deteriorate the cu The original automated Edman-Begg mulative yield in very few cycles; most in instrument, introduced in 1967, used liq struments operate at ~ 95% and can an uid-liquid extraction of an aqueous sam ple (known as "liquid-phase" sequencing); alyze 45-50 residues from 20-50 pmol of an amenable protein or peptide. a "spinning cup" feature spread the liq uids out on the chamber walls for greater However, if the peptide contains pro extraction efficiency. Next, glass fiber sup line, which is difficult to sequence by ports were introduced ("solid-phase se straight Edman chemistry, or amino acids quencing"); commercial sequencers could such as serine, threonine, and cysteine, sequence 100 pmol of sample peptide. which undergo β-elimination reactions, Then in 1980, Leroy Hood's group at the yields and sequencing certainty will be California Institute of Technology in thrown off. Currently available sequenc creased the detectability of sequenced ers provide alternative reagents and buffer amino acids to 1 pmol and improved repet conditions that can be programmed into itive yield by using gas-phase delivery of the protocol for difficult peptides, but Har TMA and TFA, both of which are highly ris says, "None of the sequencers han volatile, to permeate the glass fiber sup dles post-translational modifications auto port more efficiently. This modification matically. With phosphoamino acids, was incorporated into the Applied Bioyou have to modify the acid-base chemis systems instruments and was later try. Something like a myristylated amino adopted by other manufacturers. Six years acid, on the other hand, might be derivaago, says Harris, manufacturers began to tized but it will give an odd retention time move toward pulsed-liquid reagent deliv on the HPLC column." ery. "The idea is that you wet the sup Residual sample buffer components port with the first pulse, and you accumu can cause pH disturbances, and peptides late more material in a smaller volume immobilized on a glass filter interact less by delivering a series of microliter shots." with the glassfibersas their chain length Changes in reagent delivery and the re decreases. In the past 10 years, says Har ris, improvements in the derivatization agents themselves have reduced cycle cartridges to accommodate alternative times from the original two hours to less than an hour. The ability to queue several support materials have resulted in better resolution and higher recoveries. The use sample cartridges (with peptides stabi of PVDF membranes, brought over from lized on the support at room temperature under inert gas) for continuous overnight gel-blot techniques, and techniques for se quencing directly from a C lg cartridge, sequencing has improved the lives of introduced about five years ago by Hewlett some researchers, Harris says, and elec Packard, allow buffer salts and additives tronic or optical reagent delivery sensors that provide feedback to the controller to be washed away before sequencing. The C18 cartridge also retains the peptide take some of the worry out of running an automated instrument. Delivery times can more efficiently than glassfiberfilters do. Detection limits are in the low-picomole be programmed for individual reagents,
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Analytical Chemistry, Vol. 66, No. 24, December 15, 1994 1241 A
Product
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Reporting Experimental Data
(approaching femtomole) range, and se quencing is routinely performed at con centrations of 1-10 pmol, corresponding to a sample load of 10-20 pmol of the origi nal protein or peptide.
Selected Reprints
Analysis Considerations for HPLC and the detec tion system are generally similar to those for stand-alone HPLC systems. The PTH derivatives are generally detected at a single wavelength (usually ~ 269 nm). Di ode array detectors can be used for offpeak background subtraction. Interfering side reactions between PITC and polybrene from glass fiber filters produce com pounds that coelute with some PTH amino acids, but these can be eliminated by using other supports. The main source of uncertainty in sequence assignment comes from the increased mixture of the correct amino acids for each cycle with amino acids that failed to react or cleave in previous cycles. Current data analysis software can reduce background noise, quantitate the major peaks in each cycle, and deconvolute overlapping peaks.
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C-quencing from the other end N-terminal sequencing isn't the only way to sequence proteins; it's just the best adapted at present to automated systems. C-terminal sequencing, which uses a derivatizing reagent similar to PITC, is possi ble and in some cases preferable, as when the N-terminal amino acid of a pep tide is blocked by some modification. How ever, Harris notes that C-terminal se quencing currently demands nanomole quantities of starting peptide as opposed to the picomoles required for N-terminal sequencing and is good for only ~ 5-7 cy cles. Hewlett Packard introduced a C-ter minal protein sequencer in July. Perkin Elmer's Applied Biosystems division has an active R&D program for automated C-terminal sequencing chemistry and provides C-terminal sequencing as an inhouse contract service. "There are trade-offs," Niece says. "Ap plied Biosystems demonstrated C-termi nal sequencing on their [now discontin ued] Model 477A in 1992. Their chemis try goes 5-10 cycles but won't sequence through proline and has trouble with serine, threonine, and cysteine. Hewlett Packard's will go through proline, but it does fewer cycles." Still, he says, the pros pect of efficient C-terminal sequencing is exciting. Both he and Harris look forward to significant progress in this area over the next few years.
Automated interpretation software can make Looking forward a peptide sequence What's next for protein sequencers? "Probably better detection at low levels assignment seem using quick-reacting fluorescence-tagged reagents and fluorometry instead of UV more certain than it spectroscopy, more stable reagents, and shorter cycle times down to 10 or 20 min really is. utes," Harris speculates. Niece adds that Most researchers use the chromato graphic data to "call" the sequences them selves. Niece says that in the final re sults for a client, a stretch of four or five amino acid residues may be reported as "confident," the next may be called "tenta tive," and so on. Interpretive software is available for automated sequence assign ment, but Niece contends that pattern rec ognition programs are still not as good as an experienced researcher for calling the sequence. The software interprets the peaks in a fixed way that can produce de ceptively positive assignments even when the residue in question is labile or other wise problematic. "An experienced human knows when to be tentative about an as signment," Niece explains.
Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
both electrospray MS and CE are under consideration as alternatives to HPLC for PTH amino acid separations and detec tion, though neither is at the point of commercial development. Will MS protein sequencing replace Edman methods altogether? "I'm not con vinced that MS will ever make sequenc ers obsolete," says Harris. Data interpreta tion is still infinitely easier for Edman se quencing, although as the level of MS software improves, sequence assign ment by MS may become easier. Edman sequencers will always be significantly less expensive to own and operate than MS sequencers, and if their sensitivity reaches the femtomole range or lower, they will always be competitive with MS analyzers." Deborah Noble
