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Intact protein masses
For proteins weighing between 5 and 100 kDa, we are able to measure intact masses typically within 1 amu up to 40 kDa if the samples are sufficiently homogeneous.
Homogeneity becomes increasingly important as the molecular weight increases. About 5-10 μg are needed at a concentration of ≥ 0.5 mg/mL.
Buffers, salts, glycerol and small
detergents (such as octyl glucoside) are OK, but large detergents such as SDS or NP40 (=Triton X-100) are not.
Limited proteolysis
We often identify the stable core regions of proteins and protein complexes by limited proteolysis. The most effective way to do this is to find proteolytic conditions that
generate the species you are most interested in as the major band(s) on a gel. Relatively high protease concentrations for a relatively short time usually generate the most
useful fragments. Our favorite proteases are trypsin (for its specificity) and subtilisin (for the cleanliness of its cuts). Proteases should be deactivated with 6M
guanidine.
Peptide measurements/protein identification
These are typically either for verification of peptide structure (primary sequence) or
for protein identification by means of peptide mass fingerprinting or MS/MS. (See Protein
identification by mass spectrometry for a fuller
explanation of how this works.) These measurements are done with a MALDI ion
source and require a few hundred femtomoles of peptide. We can do in-gel and solution digests, but investigators should consider learning how to
do these on their own if they have numerous samples (See In-gel digestion procedure).
Some tips: We much prefer colloidal or conventional Coomassie stained gels to silver-stained ones (higher peptide yield). Run gels carefully
(wear gloves, etc.) to prevent keratin contamination. When excising bands, cut close to the stained part (even at the risk of losing some
protein) to exclude extra gel material. We also have an Edman protein sequencer, which is useful in some instances.
Post-translational modifications
We also identify sites of phosphorylation, acylation, glycosylation, etc. The success of these endeavors is invariably sequence-dependent and can vary from being very quick
and easy to being essentially impossible. By all means speak with us before embarking on this venture.
Peptide synthesis
We are able to synthesize peptides, typically ones that are difficult to have done commercially due to size, unusual derivatives, multiple disulfides, etc.. Commercial
companies are a better choice for routine work. We can easily check their products by MS if we have a few μg.
Antibody generation
We have considerable experience in identifying antigenic amino acid stretches within protein sequences that are tractable and can be used to generate useful antibodies.
Please consult us on this matter if you are planning this type of experiment.
Current instrumentation
ESI-FT/MS (Bruker 9.4T Qe) MALDI tof/tof (AB 4800 tandem TOF MALDI) MALDI MS (Bruker Reflex III) ESI-Ion trap (Bruker/Agilent Esquire) Edman sequencer (AB 492) Peptide synthesizers (AB 431A) Various off-line HPLCs Spectropolarimeter, fluorimeter
