A Brief History of Peptide Science, From Insulin to Today

Peptide chemistry has a compact and unusually well-documented history: a sequence of technical problems, each solved in a way that made the next one visible. This brief history of peptide science traces the main steps, from reading a sequence, through making one reliably, to understanding what sequences do and engineering them. The purpose is practical rather than antiquarian, since most of the conventions and constraints that shape a modern peptide certificate of analysis were set by these developments.

1951: reading a sequence

Until the middle of the twentieth century it was not established that a protein had a single, defined amino acid sequence. Sanger and Tuppy settled the question for one chain of insulin, publishing back-to-back papers in 1951: the first identified short peptides from partial hydrolysates, and the second worked out the order of residues in the phenylalanyl chain from enzymatic digests [1,2]. The method was laborious, reconstructing a sequence from overlapping fragments, but the conclusion was foundational: proteins are defined chemical entities, not statistical mixtures.

That result created the next problem. If a sequence is defined, it should be possible to make it.

1963-1984: making a sequence reliably

Solution-phase peptide synthesis was possible but punishing, since every intermediate had to be isolated and purified. R. Bruce Merrifield's answer was to anchor the C-terminal residue to an insoluble polymer bead, so that excess reagents and by-products could be removed by filtration and washing after every step. He set out the approach and its consequences in his 1984 Nobel lecture, including the extension to automated instruments [3].

The mechanics of the cycle are covered in our overview of solid-phase peptide synthesis. Two of its legacies are visible on every certificate of analysis:

• Purity is inherited from stepwise efficiency. Because coupling yields compound over the chain, deletion sequences are the characteristic impurity class of synthetic peptides.
• The salt form comes from the process. Trifluoroacetic acid cleavage and TFA-containing preparative HPLC leave basic peptides as trifluoroacetate salts.

Fmoc/tBu chemistry later displaced the original Boc/benzyl approach as the standard, using base-labile temporary protection paired with acid-labile side-chain groups, and modern Fmoc synthesis is what almost all catalogue peptides are made by today.

1994: joining segments

Stepwise synthesis has a practical ceiling: yields and aggregation make chains beyond roughly 50 residues difficult. Dawson, Muir, Clark-Lewis and Kent removed the barrier with native chemical ligation, joining two fully unprotected peptide segments in aqueous solution through the reaction of a C-terminal thioester with an N-terminal cysteine, which rearranges to give a native amide bond at the junction [4]. Chemical synthesis of protein-sized molecules became feasible, and with it the ability to install unnatural residues and isotopic labels at defined positions.

1990s: receptors and ligands, in that order

The same decades transformed the biology. Receptor cloning made it possible to identify a receptor before its natural ligand was known, then to go looking for the ligand.

The growth hormone secretagogue receptor is the textbook case. Howard and colleagues cloned a receptor from pituitary and hypothalamus in 1996, identified through its response to synthetic secretagogue peptides, with no endogenous ligand in hand [5]. Three years later, Kojima and colleagues isolated that ligand from stomach: ghrelin, a 28-residue peptide bearing an n-octanoyl group on Ser3, an acyl modification they reported was required for activity in their assays [6]. The sequence of events, synthetic ligand first, receptor second, natural ligand third, inverted the classical order of endocrinology and is now routine.

Receptor pharmacology concepts that came out of this era are summarised in GPCR signaling basics for peptide research.

Small molecules in the same story

Peptide science did not develop in isolation from metabolic biochemistry, and two catalogue compounds mark points in that parallel narrative.

AICAR is a nucleoside whose intracellular phosphorylated form, ZMP, mimics AMP. Henin and colleagues reported in 1995 that ZMP stimulated rat liver AMP-activated protein kinase severalfold in vitro, and that adding the corresponding riboside to isolated rat hepatocyte suspensions led to accumulation of ZMP with concentration-dependent inactivation of acetyl-CoA carboxylase and HMG-CoA reductase, inhibiting fatty acid and cholesterol synthesis in those preparations [7]. The compound became a standard pharmacological tool for probing this kinase in cell-based work, and it is discussed further in AICAR and AMPK activation research.

NAD has an even longer arc. Identified as a hydride-transfer coenzyme early in the twentieth century, it was substantially reframed once its role as a consumed substrate became clear. Belenky, Bogan and Brenner's review sets out this dual identity: NAD+ serves both as a coenzyme for oxidoreductases and as a substrate for ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins, and they describe nicotinamide riboside as a then-recently discovered eukaryotic precursor [8]. Cellular NAD+ biochemistry is covered in NAD+ in cellular metabolism research.

1990s onward: analysis catches up with synthesis

A less celebrated but equally consequential shift happened on the analytical side. Electrospray ionisation and matrix-assisted laser desorption made accurate mass measurement of intact peptides routine, and coupling liquid chromatography to mass spectrometry meant that identity and purity could be assessed in a single run rather than inferred from amino acid analysis and elemental composition. Tandem fragmentation then made it possible to confirm not just the mass of a peptide but the order of its residues.

The effect on quality expectations was immediate. Impurities that had previously been invisible, deletion sequences differing by one residue, retained protecting groups, oxidation products, became routinely detectable and therefore routinely specified. The modern certificate of analysis, pairing a chromatogram with an observed mass, is a direct product of this period, and the impurity classes it reports are catalogued in common peptide impurities.

The modern picture

The recurring themes of the present era are the ones a working lab encounters directly: stability engineering through lipidation, cyclization and unnatural residues; analytical characterisation by LC-MS as a default rather than an exception; and a steadily expanding definition of what counts as a synthesisable target.

What has not changed is the relationship between process and product. The impurity classes catalogued in common peptide impurities are the direct descendants of Merrifield's stepwise cycle, and the per-lot chromatograms and spectra published on our lab reports page exist because that cycle is never perfectly efficient.

Timeline at a glance

Year	Development
1951	Sanger and Tuppy sequence the insulin B chain [1,2]
1963-1984	Merrifield develops and extends solid-phase synthesis [3]
1994	Native chemical ligation reported [4]
1996	Growth hormone secretagogue receptor cloned [5]
1999	Ghrelin identified as its endogenous acylated ligand [6]
2000s onward	Fmoc SPPS, LC-MS characterisation and stability engineering become standard

Key takeaways

• Sanger's insulin work established that proteins have defined sequences, making synthesis a meaningful goal.
• Merrifield's solid-phase method replaced intermediate purification with washing and made automation possible.
• Native chemical ligation lifted the practical size ceiling on chemical synthesis.
• Receptor cloning inverted the classical order of discovery, as the GHS-R1a and ghrelin sequence of events shows.
• Metabolic tool compounds such as AICAR and coenzymes such as NAD+ run alongside this history and remain standard laboratory reagents.

This article summarizes published research for informational purposes. All Ascent Sciences products are for laboratory research use only and are not for human or animal consumption.

References

1. Sanger F, Tuppy H. The amino-acid sequence in the phenylalanyl chain of insulin. I. The identification of lower peptides from partial hydrolysates. Biochem J. 1951;49(4):463-481. PubMed
2. Sanger F, Tuppy H. The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates. Biochem J. 1951;49(4):481-490. PubMed
3. Merrifield B. Solid phase synthesis. Nobel lecture, 8 December 1984. Biosci Rep. 1985;5(5):353-376. PubMed
4. Dawson PE, Muir TW, Clark-Lewis I, Kent SB. Synthesis of proteins by native chemical ligation. Science. 1994;266(5186):776-779. PubMed
5. Howard AD, Feighner SD, Cully DF, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science. 1996;273(5277):974-977. PubMed
6. Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 1999;402(6762):656-660. PubMed
7. Henin N, Vincent MF, Gruber HE, Van den Berghe G. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase. FASEB J. 1995;9(7):541-546. PubMed
8. Belenky P, Bogan KL, Brenner C. NAD+ metabolism in health and disease. Trends Biochem Sci. 2007;32(1):12-19. PubMed