Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting.     

C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

Background  

Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.     

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

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.     

Validated MALDI-TOF/TOF mass spectra for protein standards

A current focus of proteomics research is the establishment of acceptable confidence measures in the assignment of protein identifications in an unknown sample. Development of new algorithmic approaches would greatly benefit from a standard reference set of spectra for known proteins for the purpose of testing and training. Here we describe an openly available library of mass spectra generated on an ABI 4700 MALDI TOF/TOF from 246 known, individually purified and trypsin-digested protein samples. The initial full release of the Aurum Dataset includes gel images, peak lists, spectra, search result files, decoy database analysis files, FASTA file of protein sequences, manual curation, and summary pages describing protein coverage and peptides matched by MS/MS followed by decoy database analysis using Mascot, Sequest, and X!Tandem. The data are publicly available for use at ProteomeCommons.org.