Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures. Copyright © 1998 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35074/1/419_ftp.pd

Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to rapidly detect and profile large proteins from Escherichia coli whole cell lysates in the mass range 25–500 kDa. The bacterial samples were treated with guanidine hydrochloride and Triton X-100 to disrupt and solubilize the large inner membrane proteins. A sample preparation involving a nitrocellulose polymer film, and α-cyano-4-hydroxycinnamic acid, sinapinic acid or caffeic acid as matrix was utilized to rapidly monitor the presence of induced and repressed protein synthesis in response to l -arabinose catabolism in E. coli cells. The results were compared to those of 1-D or 2-D gel electrophoresis. © 1997 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35070/1/95_ftp.pd

Multidimensional analyses of the proteome using nonporous reversed-phase HPLC coupled with MALDI -TOF and ESI mass spectrometry.

This thesis work consists of four major parts: solubilization of large proteins, profiling of large proteins from whole cell lysates, the use of nonporous (NP) RP-HPLC columns for protein separation, and the development of multidimensional nonporous LC-MS methods. In order to gain a better understanding of the biological system, new techniques must be developed to complement 1-D and 2-D gel electrophoresis methods. The use of a nonionic detergent (either Triton X-100 or n-octyl glucopyranoside) in the lysis buffer, along with guanidine-hydrochloride, has significantly improved the solubilization of large proteins (>>50 kDa). Using these procedures, proteins over 100 kDa have been profiled from E. coli extracts with MALDI-TOF MS. NP-RP-HPLC has been used to separate proteins up to 90 kDa from human breast cancer and Siberian Permafrost bacterial whole cell lysates with sufficient resolution for subsequent MS or enzymatic digest analysis. The C18 coated 1.5 mum diameter spherical NP packing materials provide a uniform surface that prevents proteins from sticking inside the pores. This means improved protein recovery (up to 80%) and separation time (15--30 min). Oncoproteins implicated in human breast cancer, including C-src, P53, and Hsp 27, have been identified from various MCF10 cell lines. In addition, Csp C, a cold shock protein from Permafrost 7-3 strain, has also been isolated. The 1-D image generated from the on-line NP-RP-HPLC-ESI-MS method, therefore, provides a direct means of comparing the highly expressed proteins in different samples or at different stages of tumorigenic progression. The development of chromatofocusing NP-RP-HPLC-ESI-MS method, as an alternative, allows for rapid separation of proteins based upon pI and hydrophobicity in the first and second chromatographic dimensions. Molecular weight (MW) is determined on-line in the third dimension using an ESI-MS. The resulting 2-D protein image is analogous to a 2-D gel image, pI vs. MW along with abundance information. This technique can be fully automated as well. Consequently, it is a practical alternative to the more time consuming and labor-intensive 2-D gel methods in the study of proteomics.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132760/2/9990868.pd