ZORBAX Strategy for Reversed-Phase Method Development of Proteins and Peptides

Additional Method Development Recommendations
Proteins and Peptides Using Reversed-Phase LC/MS Methods

Method Development of Proteins and Peptides

This ZORBAX Column Selection Strategy provides some hints on method development for proteins and peptides. For small peptides, Molecular Weight < 2000 please click here to view the method development strategy. For method development of larger peptides and proteins review the suggested guidelines outlined below. Efficient reversed-phase separations of large molecules require columns with a wide-pore size (300Å).

Choose the Initial Column and Conditions for Proteins and Peptides
Peptides, Polypeptides, Proteins (MW < 50 kDA)	Peptides, Polypeptides, Proteins (MW < 1,000 kDA)
StableBond 300SB-C8 Initial Bonded Phase Choice Poroshell 300SB-C18
300SB columns are wide-pore columns with unbeatable lifetime in TFA containing mobile phases. This makes them an ideal first choice for separations of peptides and proteins. C8 is an excellent starting bonded phase because of its moderate hydrophobicity C18 and C8 are generally selected for peptides and protein digests but can also be used for proteins C3, C4 and CN are generally selected for larger, hydrophobic polypeptides and proteins but can also be used for peptides.	Poroshell 300SB columns use an innovative particle technology to deliver rapid protein separations. Short analysis times with efficient peaks are easily obtained with Poroshell columns. Using the 300SB bonded phases also gives these columns exceptional lifetime with TFA containing mobile phases. C18 is a good starting bonded phase choice for most peptides, polypeptides and proteins to maximize retention C8 is generally selected for moderate size proteins but can be used with any sample C3 is generally selected for antibodies or large proteins but can be used for any sample
Initial Separation Conditions
Column: 300SB-C8 4.6x150 mm, 3.5 or 5µm Part Number: 883995-906 Part Number: 863973-906 Mobile Phase: A: 95% H₂O:5% ACN with 0.1% TFA B: 5% H₂O:95% ACN with 0.085% TFA Gradient: 0 – 60% B in 60 min Temperature: 35 - 40ºC Flow Rate: 1 mL/min	Column: Poroshell 300SB-C18 2.1 x 75mm, 5µm Part Number: 660750-902 Mobile Phase: A: 95% H₂O:5% ACN with 0.1% TFA B: 5% H₂O:95% ACN with 0.085% TFA Gradient: 0 – 60% B in 10 min Temperature: 35 - 40ºC Flow Rate: 2 mL/min
Start at low pH with simple aqueous / organic gradient Typically a water/acetonitrile with 0.1% TFA gradient is used to elute all components of interest. A typical high-resolution gradient on a 300Å pore size column requires 30-60 minutes. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptional resolution. Then to improve resolution, increase the gradient time, decrease column length, or increase flow rate.
Optimize sample solubility For best peak shape and recovery at any pH, it is important to solubilize a sample completely. Highly acidic or neutral solvents can be used with ZORBAX 300StableBond and Poroshell 300SB while neutral solvents and dilute bases can be used with ZORBAX 300Extend-C18.
Solvent Choices to Solubilize Proteins and Peptides Water/phosphate buffer Weakest Dilute acid (TFA, Acetic Acid or HCl) Neutral pH, 6-8M Guanidine-HCl or isothiocyanate to 5% HOAc/6M Urea Dilute acid + aqueous/organic solvents (ACN, MeOH, THF, IPA) Dilute Base (Ammonium hydroxide) DMSO or 0.1%-1% TFA in DMSO Strongest Formamide

Raise the Temperature

Separations of proteins and peptides are influenced by temperature and higher column temperature can dramatically improve both resolution and recovery of proteins and hydrophobic and aggregating peptides.

StableBond 300SB – up to 80ºC Poroshell 300SB – up to 80ºC

Optimize Mobile Phase pH
Try mid and high pH if low pH does not work

If an optimized low pH method does not provide an ideal separation, then mid or high pH mobile phase can be used. At high pH selectivity is often very different because acidic amino acids become negatively charged and some basic amino acids may loose their charge. ZORBAX 300Extend-C18 is an excellent choice for mid to high pH separation.

Column: 300Extend-C18, 4.6 x 150 mm, 5 mm
Part Number: 773995-902

Mobile Phase: A: 20 mM NH₄OH in H₂O
B: 20 mM NH₄OH in 80% ACN

Flow Rate: 1 mL/min
Temperature: 25 - 30ºC (not >60ºC)
Gradient: 5 – 60% B in 30 minutes

Starting Column Choices for Analytical Separations of Peptides and Proteins

Low pH

Mid and High pH

MW < 50 kDa
300SB-C8
4.6 x 150 mm, 3.5µm
Part Number: 863973-906

MW < 1000 kDa
Poroshell 300SB-C18
2.1 x 75 mm, 5µm
Part Number: 660750-902

MW < 25 kDa
300Extend-C18
4.6 x 150 mm, 3.5µm
Part Number: 763973-902

Additional Method Development Recommendations
Proteins and Peptides Using Reversed-Phase LC/MS Methods