Practical Protein Chemistry - A. Darbre 1989

Current state of automated liquid-phase amino acid sequence analysis
Sample preparation

It is impossible to discuss Sample size and suitability for analysis without addressing the specific features and capabilities of the instrument itself, the selected analytical program, and the auxiliary equipment, as the direction of the Structure/133.html">Discussion is largely dictated by whether a 1 M or 0.1 M quadrol solution [3] is used, or whether Polybrene is employed with or without a refrigerated trap. In most cases, depending on these differences, operational parameters can vary by nearly an order of magnitude (though not more). Furthermore, The Nature of the buffer (quadrol, N,N-dimethylallylamine, or DMAA for short) significantly affects the determination results.

Thirteen years ago, when our laboratory was determining the Amino acid sequences of immunoglobulin heavy and light chains, establishing a sequence of 30–40 Amino Acids typically required 10–20 mg of sample. Today, using a modified instrument, 0.1 M quadrol, polybrene, and a refrigerated trap, we routinely determine 60–80 residues using 1–2 mg of sample loaded into the instrument (a two-fold increase in Cleavage efficiency). It is difficult to pinpoint the exact reasons for this improvement; most likely, it is the result of the combined influence of many factors.

Typically, we prepare samples for Sequencer analysis by lyophilizing them from Water, dilute acid solutions, or volatile aqueous buffers. The samples are stored in sealed scintillation vials as a fluffy white powder. In our practice, we have occasionally encountered samples lacking this appearance that could not be analyzed. The reasons for such failures remain unclear, but they may be related to sample insolubility and the presence of impurities. Due to the challenges of identifying Cysteine residues by most conventional Methods for PTH-Amino Acid Derivatives, we almost always reduce and carboxymethylinate our samples with radioactive Reagents prior to sequence analysis. Consequently, a small aliquot of each PTH-derivative is checked for radioactivity.

Salt-containing samples must not be loaded into the sequencer Reactor, as this can lead to the elution of the entire sample. This is easily observed when analyzing radiolabeled preparations, where radioactivity in the products of the first two or three cleavage cycles drops sharply and continuously, eventually reaching Background levels. Therefore, the presence of salts in the sequencer reactor should be avoided. In recent years, samples are frequently loaded in the presence of SDS. Although this approach is not widely used in our laboratory, the presence of up to 1% SDS is generally beneficial for dissolving samples, particularly Membrane Proteins isolated from SDS gels, without compromising subsequent Cell/13.html">Protein Structure determination.

There is no consensus on the optimal solvent for sample loading into the reactor, and solvent systems vary from one laboratory to another. Apparently, this issue has never been systematically investigated. We dissolve our samples in 25% acetic acid. If they fail to dissolve, they are heated to 80 °C for 10–30 minutes while thoroughly mixed on a vortex mixer. If this Treatment also fails, the sample is immediately lyophilized and stored in that state for 24 hours. The following day, two alternative dissolution pathways are available. First, one can attempt dissolution in 1 M ammonia. Many proteins dissolve at a higher pH, so switching to 1 M ammonia brings about half of the proteins insoluble in 25% acetic acid into solution. If the protein remains insoluble in ammonia, it is usually lyophilized again, and sequencer loading is delayed for another 24 hours. A second approach, frequently used in our laboratory, involves dissolving the sample in a concentrated volatile organic acid (formic or heptafluorobutyric acid). Heptafluorobutyric acid (HFBA) is routinely used for this purpose in many laboratories. We avoid it due to the difficulties associated with handling, storing, and using HFBA as a general-purpose solvent. However, one can apply the acid introduced into the sequencer, thereby eliminating some of the complications associated with storing this hazardous reagent.

Proteins known to possess an unblocked N-terminal amino acid that nevertheless fail to yield results in sequencer analysis do so for various reasons. If the pH (>7.0) and alkylating agent concentration were not rigorously controlled during reduction and alkylation (in guanidine hydrochloride or urea), There is a distinct possibility that the alkylating agent reacted with the N-terminal amino acid. When this happens, the protein becomes "blocked." This specific issue is a disaster, as there is no convenient way to remove the alkylating group. A second difficulty arises when a protein is insoluble in one or more reagents (Solvents). We have found that this most frequently applies to the quadrol buffer. Often, when analysis was impossible in the presence of quadrol, replacing it with a DMAA buffer yielded excellent results.

Following the loading of each sample, several operations must be performed prior to THE START OF each analytical run on the instrument. Two such operations are routinely conducted in our laboratory. The first is "sample drying," which involves alternating evacuation and flushing of the reactor with argon. This preparation yields a thin, uniformly distributed dry film of the sample on the reactor wall. This step is performed automatically before each analysis, but is then automatically excluded from the main analytical program. This is typically followed by a second preparatory step: one complete cleavage cycle with the delivery of FITC to the reactor turned off (the delivery valve for reagent R₁ in the "off" position). This delays the overall determination process by no more than 10 minutes following sample drying, because immediately after the program executes the "add R₁" command, the "add R₁" valve can be manually engaged, allowing the entire process to proceed automatically According to the standard routine.

Microprocessor programming simplifies the execution of these operations. The rationale behind the aforementioned "drying–evacuation" step is obvious. Prior to adding FITC and quadrol, it is crucial to thoroughly dry the sample and distribute it evenly across the reactor surface. Typically, samples are analyzed without The addition of FITC During the first cleavage cycle to wash away any extraneous impurities readily detectable by HPLC. This preliminary sample treatment serves as an additional means of standardizing the sample loading conditions and preparing it for analysis.