Quality Control

Quality Control Equipment:

HPLC – Gilson HPLC, Agilent UHPLC

Mass Spectral Analysis –Bruker Microflex LT MALDI-TOF, Bruker amaZon SL ion trap

Amino Acid Analyzer – Waters Acquity UPLC H-Class

Reagent Preparation:

HPLC Buffers:   A: HPLC grade water with 0.1% TFA; B: Acetonitrile with 0.08% TFA

Mass Spectral Matrix:   10mgs 4 hydroxy-alpha cyano cinnamic acid in 500uL A, 500uL B

Dissolve in Eppendorf, spin down pellet, decant and use supernatant.

Quality Control Protocol:

1. HPLC Analysis
   HPLC Injection:  approximately 1mg/mL peptide in HPLC grade water, 100uL (100ug) per injection
   UHPLC Injection: approximately 1mg/mL peptide in HPLC grade water, 1uL (1ug) per injection
   * Other solubility techniques may be required for hydrophobic sequences*
   Percent purity based on peak area.
2. Mass Spectral ESI
   Injection:  approximately 1mg/mL peptide in HPLC grade water, 1uL (1ug) per injection.
   * Other solubility techniques may be required for hydrophobic sequences*
   Calculated Mass within 0.1% of Molecular Weight.
3. Mass Spectral MALDI-TOF
   Spot 1uL matrix with 1uL /mL peptide solution.
   Let air dry, run sample.
   Different voltages, ion charges, and setting shown on Mass Spectral Analysis.
   Calculated Mass within 0.1% of Molecular Weight. (Note: if MW is less than 2000 daltons, MW to be within 2 daltons.)
4. AAA Analysis (If required)
   Experimental results of the sample must be within 20% for acceptable amino acids
   Sequence ratios must be confirmed by analysis
   Note: Serine and threonine are partially destroyed during hydrolysis.  Cysteine, methionine, and tryptophan residues are destroyed during hydrolysis.  They will not appear in Amino Acid Composition.  The peptide hydrolysis process converts Asn (N) to Asp (D) and Gln (Q) to Glu (E).  Thus, N and Q residues are calculated theoretically as D and E residues, respectively. 
5. Dilutions (If required)
   Original stock solution concentration was used to determine dilution factor.  A dilution was then made to requested concentration.  If duplicate/triplicate AAA is requested, an average concentration is used to determine dilution factor.

Reassay Interval of Stored Samples:

Every 6months or each time a lot is aliquoted from bulk storage.

Molecular formula and mass shifts for common 13C and 15N universally labeled amino acids.