Delving into the Unknome: Purification of Peptides and Proteins

Delving into the Unknome: Purification of Peptides and Proteins

Hello, and welcome to the “Colorful Researchers” blog. I’m Padma, one of the chromatography specialists here at Buchi Labortechnik. I, along with my colleagues Peter (Evaporation) and Bruno (Freeze/Spray Drying), have been given the difficult task of following on from Bart, who had over 35 years of experience in the field of chromatography and purification. Sadly, Bart has decided to hang up the mantle, so it’s up to us to do our best to continue his mission of sparking “the truly magnificent and idiosyncratic curiosity of the researcher”. We all wish Bart the very best in his retirement and thank him for his contribution to science and the advancement of laboratory technology and techniques.

The Importance of Proteins and Peptides

The subject of today’s chromatography blog is proteins & peptides, which have fascinated me since I was first introduced to amino acids at the University of Mumbai. Although there are over 500 amino acids, only 22 appear in the genetic code of all life. These α-amino acids are the building blocks of peptides and proteins, one of four fundamental classes of macromolecules in nature. I always thought it was miraculous how so few building blocks could be used to create such varied, highly organized three-dimensional structures that are closely related to their biological function.

Amino Acid                  Peptide                        Protein

Proteins and peptides are responsible for various processes critical to sustaining life. Proteins often act as messengers to transmit signals within a cell in response to external stimuli. They also play a role in cell growth, division, and differentiation. Some proteins act as transcription factors, influencing which genes are turned on and off. They act as enzymes to speed up chemical reactions, and proteins like keratin (in hair and nails) and collagen (in skin and connective tissue) provide structural integrity. Hemoglobin is a protein in blood that transports oxygen. Antibodies are proteins that recognize and neutralize foreign substances. Actin and myosin are proteins responsible for muscle contraction, and cadherins and integrins help cells stick together. Proteins and peptides are like the governing body governing our bodies – handling transport, defense, infrastructure, communication, energy, the list goes on, and that is only talking about what we know. Time to delve into the unknown!

The Unknome Database

The human genome encodes ~20,000 proteins, many of which are still uncharacterized. Due to the vast number of poorly understood proteins, the “Unknome database” project has been established to rank proteins based on how little is known about them. The existence of such a database highlights the need for more research, which means more chromatography!

“Proteins and peptides are fundamental to biological processes in living cells, and the human genome encodes ~20,000 proteins, many of which are still uncharacterized.”

We have a phrase in India: सौ मार्ग एक ही लक्ष्य के लिए।, which translates as “A hundred paths lead to the same goal” – similar to the English saying, “All roads lead to Rome”. The same is true for the separation and purification of proteins. Several techniques, including crystallization, filtration, precipitation, or chromatography, can perform the purification step. However, chromatography is by far the most commonly used method for high-resolution target compound purification.

Purifying Proteins and Peptides using Chromatography

Numerous chromatographic matrices have been developed since the 1950s to separate proteins and peptides. These matrices are used in the liquid chromatographic techniques in the table below:

Although there are many paths to the same goal, or roads leading to Rome, some paths are less arduous than others. With regard to chromatography, reversed-phase (RP) chromatography has become an essential tool for small protein and peptide purification on a larger scale for investigative studies. The separation power of RP chromatography enables the separation of polypeptides that differ by only a single amino acid. The method is also compatible with aqueous samples, as water mixtures are used as the mobile phase.

When considering RP chromatography, there are two techniques to consider: flash and preparative high-pressure liquid chromatography (prep HPLC). Flash is generally used as a pre-purification step to purify large amounts at a reasonable resolution, whereby prep HPLC is used to achieve the highest resolution (purity).

As shown in the table above, the particle size influences the pressure required to perform the separation. Prep HPLC columns require a high-pressure pump to get the mobile phase through the column due to the large surface area created by the smaller particle sizes. The pressure generated is influenced by several parameters indicated by the following equation.

ΔP: change in pressure (bar, psi)

F: flow rate (mL/min)

L: column lengths (mm)

K: specific column permeability

dp: particle size (μm)

r: column radius (mm)

n: solvent viscosity (cP) (1 for water, 2 for water/ MeOH 50/50)

The primary factors influencing pressure are column radius and particle size. The pressure values are inversely proportional to the square of particle size and column radius; therefore, if the particle size is halved, the pressure must be increased by a factor of four.

Tips & Tricks

When considering Flash and prep  HPLC, from my experience with chromatography, I would recommend using flash chromatography as a first step to pre-purify as much of the target compound as possible. Then, I would use prep  HPLC on the collected flash fractions to achieve the desired resolution. Doing so frees the injected material from most contaminants in the pre-purification step.

The Essence of Polarity

The crux of any separation is polarity, and for our peptides and proteins to be separated efficiently, they must find a sense of camaraderie with the stationary phase. To put it simply, if the compounds don’t “gel well” with the stationary phase, they will not be separated. Each of the stationary phases has its own temperament. C18 is the most introverted (or hydrophobic), while C4 is more sociable; with its inclination for company, it retains those more sociable (polar) molecules.

The Dance of Separation – The Mobile Phase

Solvents play a significant role in the grand dance of separation and get things moving. Water typically plays a leading role with its guest star, often the versatile acetonitrile, known for its flowing moves (low viscosity), not hogging the spotlight (low UV detection), and the ability to gracefully exit the stage post-separation (easily removed or evaporated after the separation process). The choreography of this dance involves a gradient, a gradual crescendo of the less polar solvent (that elutes first), culminating in sharp peaks and a high-resolution performance. And, of course, how could I forget the supporting role played by trifluoroacetic acid? Present in tiny amounts (about 0.1%), it ensures the pH stays in tune (buffering) and boosts the molecules’ performance with some ion-pairing magic that enhances retention.

The Grand Stage – Sample loading

Every dancer - or molecule - needs the right stage, and for the more extroverted (polar) proteins and peptides that don’t prefer the C18 stage, we must roll out the C8 and C4 carpets. The size of the stage (pore size) is pivotal. While cozy 60-100Å stages are perfect for petite molecules, such as peptides, the grand 200-300Å arenas are ideal for the more significant and more substantial protein stars. Remember that for larger-than-life proteins that exceed 5kDa, the 200-300Å stage is a must.

By following these tips and tricks, proteins and peptides can be purified to a high degree and will be ready to be concentrated  for subsequent analysis. Temperature-sensitive compounds can be spray-dried without significant loss of activity. Bart previously spoke about whether to freeze or spray-dry peptides and proteins and the effects of freeze /spray-drying on peptides. For all other compounds, rotary evaporation  is frequently used for solvent removal.

So, there you have it, my dear readers! The mesmerizing art, science, and dance of purifying proteins and peptides, wrapped up in a brief guide. Here’s to many more successful separations, and until next time, stay curious and keep experimenting! 🧪🔬🌟

Phir Milenge Chalte Chalte,

Padma