A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Abstract

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

“…A saturated CHCA in 50% acetonitrile and 1% TFA was diluted to a 1:4 ratio (v/v) in 50% acetonitrile and 1% TFA. Angiotensin I, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH (18-39) were added to the 1:4 diluted matrix solution. The resulting matrix-standard solution was vortexed.…”

“…They also have the drawback of embedding the proteins of interest in a polyacrylamide gel, which necessitates awkward recovery and purification steps if subsequent analytical methods are to be applied downstream. In order to overcome these difficulties we have developed a 2-D liquid phase protein separation method that greatly facilitates the mapping of the protein content of human cells [10,11]. The method is based upon pI separation in the first dimension and non-porous RP-HPLC in the second dimension.…”

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

“…Instead, optimizing the average resolution (R avg ) for all peak pairs is much more important. The experimental average resolution for a mixture of n components is defined by: (8) By inserting the fundamental definition of resolution for a given pair of solute i and i+1 into eq 8 (t R and W are the retention time and baseline peak width): (9) and assuming a constant peak width of W avg under gradient conditions, we can show that: (10) By comparing eq 10 with eqs 6 and 7, it is clear that the average resolution is proportional to the peak capacity computed via eq 7: (11) Thus, optimizing the peak capacity (n c **) is equivalent to optimizing the average resolution. In this paper, we first study the effect of the individual operational variables (gradient time, flow rate, temperature and final eluent strength) on the peak capacity (n c **) of peptides for a fixed column format (i.e.…”

Peer Reviewed: Top-Down Proteomics

“…For bacteria, Fenselau's team showed that identification of small B. cereus proteins could act as potential markers for infectious spores (e.g., anthrax; 22). Lubman and co-workers have applied isoelectric focusing and RPLC, ESI/TOF, and offline tryptic digestion to identify proteins and PTMs from human cancers (49). Together, these approaches hold the promise of nearly 100% reliability (high predictive value) for earlier detection of complex diseases or drug efficacy.…”