Methods and analytics

How mass spectrometry confirms that a peptide is the intended sequence: electrospray and MALDI, working out the expected mass, reading charge states and adducts, sequence confirmation, and the limits of the method.

Where wrong-configuration residues come from in synthesis, why mass spectrometry and HPLC can miss them, and how hydrolysis in deuterated acid and chiral separation measure them.

How a peptide is broken apart inside the mass spectrometer, how the b- and y-ion ladders read out its sequence, which fragmentation methods suit which questions, and what MS/MS cannot settle.

How reversed-phase HPLC measures the chromatographic purity of a peptide, what the figure includes, and what it leaves out.

What a peptide certificate of analysis should contain, how to check that it belongs to the material in hand, how to read its identity and purity results, and the signs that it deserves a second look.

What the impurity table on a certificate lists, how relative retention times, the largest single impurity and total impurities fit together, what the thresholds mean, and how to read it.

Water, counter-ions, salts, residual solvents, scavengers, plastics, metals and endotoxin are in the vial but not in the purity chromatogram: why each is invisible at 214–220 nm, how each shows up on a mass spectrum as adduct ladders and repeating series, and the methods that measure them.

Why a peptide that is 99% pure by HPLC can be only 75–85% peptide by weight: what the powder contains, how each part is measured, and how the two figures combine.

Why synthetic peptides are salts, where trifluoroacetate comes from, when it matters in the laboratory, and how it is exchanged for acetate or chloride.

How hydrolyzing a peptide and counting its amino acids confirms its composition and measures how much peptide a sample contains, and what the method cannot see.

How Karl Fischer titration measures the water in a freeze-dried peptide, why the coulometric and oven methods suit peptide samples, what interferes, and how the result is used.

Solid-phase synthesis from the first residue to the freeze-dried powder: protecting groups, the coupling cycle, cleavage, purification, and where each kind of impurity comes from.

What endotoxin is, why traces of it confound cell-based in vitro assays, how it gets into synthetic peptides, how it is measured, and what makes the measurement wrong.

How peptides degrade, chemically and physically, why the dry solid lasts far longer than a solution, which sequences are most at risk, and the storage and handling that slow it down.

From a freeze-dried vial to a solution of known concentration: the arithmetic, the solvent, the charge chemistry and the handling, with the errors each step can introduce and the article that covers it.

The equation that turns a mass of peptide into a molar concentration, which molecular weight to use, a worked example from a certificate, and how to check the result by absorbance.

What each concentration unit means, how to convert between mass per volume and molarity, where the factors of a thousand hide, and conversion tables for the figures a laboratory meets most.

Why a concentrated stock is made first, the dilution equation, serial dilutions and how their errors compound, which volumes a pipette is good at, and what to dilute into.

Balance resolution and minimum weight, static and water uptake, the size of each error for a few milligrams of powder, and the whole-vial method that avoids weighing altogether.

How to read a sequence for charge and hydrophobicity and choose a first solvent, the chemistry of dilute acid and base, DMSO and the other organic co-solvents, chaotropes, and how much of each is too much.

A troubleshooting guide: the powder that floats, the solution that clouds, the gel, the yellow tint and the overnight precipitate, each traced to charge, hydrophobicity, aggregation, oxidation or the material's history.

The ionizable groups and their pKa values, the Henderson–Hasselbalch equation, the isoelectric point and why solubility is lowest there, and how to choose and make a buffer with enough capacity.

What the two preserved aqueous reagents contain, in every unit, what 0.9% benzyl alcohol does to microbes and to peptides, and the spectroscopy, chromatography and cell-based work where plain water is the better solvent.

What happens to a peptide in solution over time, the single-use aliquot habit, temperature and frost-free freezers, which containers adsorb the least, light and oxygen, and when to re-check a stock.