Mass Spectrometry and HPLC

Biomolecular Mass Spectrometry

Both the QSTAR Elite and HCT Ultra mass spectrometers are equipped with electrospray sources, the QSTAR Elite is also equipped with a nanospray source. These sources can be used to produce either positive or negative ions. Samples are generally dissolved at concentrations of 0.2 to 0.5 micrograms/microliter in a solution of a dilute (0.1-1.0% v/v) volatile organic acid in water. The acids commonly used are acetic, formic or trifluoroacetic acid. Often an organic solvent such as methanol or acetonitrile is added to increase the sensitivity and the degree of ionization. The presence of detergents will usually completely suppress ionization, and thus prevent the collection of any useful data!

Precise molecular weights can be determined for proteins (including glycoproteins) and peptides by mass spectrometry. Mass spectrometry is particularly powerful for the identification of post-translational modifications. The specific location of the modifications can be determined by on-line HPLC/mass spectrometry of a proteolytic digestion mixture of the protein. Eluted peptides can be identified by comparison of their observed masses with the calculated masses of the predicted proteolytic peptides, if the protein sequence is known. Any observed peptide masses with unmatched calculated masses are candidates for the post-translationally modified peptides.

Samples may be introduced into the mass spectrometer by (1) capillary, (2) direct syringe pump infusion or by (3) on-line capillary or nano HPLC. The HPLC method is slower, but it provides a means of desalting and it can be used to better analyze complex mixtures.

Mass spectrometry of peptides is generally easy, proteins are less predictable. Unfortunately, not all proteins will produce data. The larger the molecule the more difficult. Small molecules (<20000 daltons) are easy and a wide range of solvent and instrument parameters will result in good data, while larger proteins are more difficult. Larger proteins only yield data in a very narrow range of conditions, and those conditions are determined mostly by trial and error. The degree of difficulty is related to factors such as protein solubility at acidic pH, the protein’s isoelectric point, and the number of disulfide bonds.

Information regarding protein identification by proteolytic digestion and HPLC/MS/MS can be found here.

Please contact the facility at if you have any questions or need help choosing a service to fit your experiment.

Current OUHSC Pricing

Service Price
MS Analysis  
Mass determination by MS for molecules <15 kDa $38.00/sample
Mass determination by MS for molecules >15 kDa $50.00/sample
MS(n) $70.00/sample
HPLC Analysis  
HPLC (UV/VIS) $75.00/sample
HPLC (UV/VIS) with Fraction Collection $85.00/sample
HPLC method development $100.00/hour
HPLC/MS Analysis  
HPLC/MS $100.00/sample
HPLC/MS (Batch of 10 samples) $750.00/batch
HPLC/MS/MS $110.00/sample

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