Analytical
Instrumentation
A consultation with Maria Person or Stony Lo is required before sample submission.
The following rates are applicable to CRED members and UT-Austin customers.
A. HPLC Analysis
Standard Analytical HPLC |
$10.00 |
HPLC-EC, 8-oxo dG Analysis |
$15.00 |
B. Mass Spectrometric Analysis
LCQ Analyses |
|
ESI-MS (per injection:MW determination of simple chemicals, peptides, proteins) |
$45.00 |
HPLC-UV/MS (per injection: microbore HPLC with UV and/or MS detection) |
$60.00 |
HPLC-ESI-MS/MS (per injection: protein identification) this includes microbore HPLC separation, MS/MS sequencing of protein and database search with TurboSEQUEST |
$120.00 |
Microspray-ESI |
inquire |
MALDI analyses |
|
MALDI-MS + MS/MS (per sample: for protein identification or peptide characterization) |
$60.00 |
MALDI-MS (per sample: for MW determination of simple chemicals, peptides, proteins) |
$45.00 |
LC-MALDI-MS/MS |
$120.00 |
C. GC-MS Analysis
Trace GC-MS (per sample: including derivitization and analysis) |
$35.00 |
Notes:
8-Oxo-dG Analysis
- The minimal quantity of total DNA required per sample is 5µg; preferred quantity is between 10 and 50 µg per sample. Dried DNA samples should be submitted in 1.5-ml microfuge tubes with quantity indicated. If DNA samples have to be in solution, please indicate the concentration and the volume of each sample (the protocol requires 10 µl of DNA solution in each sample).
- The limit of 8-Oxo-dG detection is about 30 to 50 fmol.
Mass Spectrometric Analysis
- Simple chemical - Determination of molecular weight by LC-MS, GC-MS; quantitation by GC-MS
- polar compounds can be analyzed by Electrospray Ionization (ESI) or MALDI MS
- the sensitivity of detection depends upon the ease with which each compound can be charged or protonated, the limit of detection is in sub-picomlar range.
- nonpolar compounds can be analyzed by Atmospheric Pressure Chemical Ionization (APCI) MS, with less sensitivity.
- Both polar and nonpolar chemicals can be derivatized then analyzed by GC-MS. Many compounds can be detected and quantitate in the low pg range using selected ion mode.
- Determination of molecular weight of peptides and small proteins
- ESI MS analysis can analyze intact proteins up to MW~80,000. MALDI-MS can analyze proteins >100,000. However, in order to obtain the exact molecular weight of proteins, we require high purity samples or separation by HPLC prior to analysis. As the molecular weight or impurities (including buffer adduct ions like sodium) increases, the pattern of the protein ions (protein envelop) gets less distinct and the standard deviation for the calculated molecular weight is greater. MALDI can measure molecular weights of larger proteins but requires proper calibration for good mass accuracy.
- MALDI-MS with internal calibration can measure peptide MW with 30 ppm mass accuracy.
- Protein identification:
- Peptide mass mapping: In-gel digest samples from 1D-or 2D gel electrophoresis (see Molecular Biology core for more information) can be identified using peptide mass mapping with the MALDI-MS. The masses of the tryptic digest peptides are queried against the theoretical digests of proteins obtained from a database such as UniProt and the best match is reported. When fewer than 5 matching peptides are found, MALDI-PSD fragmentation is used to identify the protein via the sequence of individual peptides as described below. The sensitivity of this approach for in-gel digest samples is roughly 100 ng, with 10x higher sensitivity for solution digests.
- Peptide Fragmentation: Measurement of the molecular weight of a protein is insufficient to provide definitive identification. The MS/MS function of the LCQ is capable of obtaining a spectrum of the fragmentation pattern for each digested peptide being analyzed after HPLC separation. Each of the MS/MS patterns is analyzed by the TurboSEQUEST search engine for the best match to a peptide derived from the protein database. Once a protein match is obtained, the peptide sequences are provided. The sensitivity of the LC-MS/MS approach is within the low microgram range, while the 100 ng range can be achieved by coupling LCQ to capillary chromotography, with even more emphasis on purity. Note: amount of protein in a gel is always greater than amount of peptides extracted after in-gel digest. Only proteins present in the database (NCBI or Uni-Prot) or highly homologous to those in the database will be identified.
