Studying proteomes is of central interest in life sciences, because expression and degradation of proteins are highly dynamic processes, which admit cells to adapt to changing conditions like drugs, temperature changes etc. In this respect, e.g. the discovery of proteins with unknown functionality or the investigation o fmetabolic pathways are main topics

Baessmann, C.

Lubeck, M.

Rabus, R.

Schweiger-Hufnagel, U.

Shen, M.

MPG.PuRe

THEAPPLICATIONSBOOK  September 20032PharmaceuticalRibosomal proteins from strain EbN1 were disrupted with thePlusOne Sample Grinding Kit (Amersham, Freiburg, Germany), andcell debris was removed by centrifugation (30 000  g, 30 min).The supernatant was subjected to an ultra-centrifugation at250 000  g for 2 h. The pellet was resuspended and  furtherpurified by centrifugation through a sucrose cushion (19 h,900 000  g) yielding in a pellet of highly enriched ribosomes. Toremove ribosomal RNA proteins were extracted with phenol. Thepurified ribosomal proteins thus obtained were reduced with DTTand alkylated with vinylpyridine prior to digestion with trypsin(Roche) following standard protocols. The other protein digestswere performed according to  standard in-solution digest protocols. 2D HPLCIon-exchange chromatography: The digested protein mixturewas bound to a strong cation exchange column (BioX-SCX 500µm, 15 mm) at pH 2.5. Salt plugs of increasing concentrationswere injected applying a step gradient, which makes MS-data setseasier to handle during post-processing. Nine peptide fractionswere eluted (Microflow, 30 µl/min with 125 µm capillaries) forfurther separation by RP-chromatography.Trapping peptides and desalting: The trapping column wasused for concentrating and desalting the eluate from the IEX. Theuse of microflow (30 µl/min with 125 µm capillaries) allowed fastsalt steps in the first dimension, and large sample volumes. WhenProtein Mixtures Using Two-DimensionalChromatography and Ion Trap MSMarkus Lubeck, Ulrike Schweiger-Hufnagel and Carsten Baessmann, Bruker Daltonik GmbH, Bremen, Germany,Ralf Rabus, Max-Planck-Institute for Marine Microbiology, Bremen, Germany, Maryann Shen, Bruker Daltonics Inc., Billerica, Massachusetts, USA.IntroductionStudying proteomes is of central interest in life sciences, becauseexpression and degradation of proteins are highly dynamicprocesses, which admit cells to adapt to changing conditions likedrugs, temperature changes etc. In this respect, e.g. the discoveryof proteins with unknown functionality or the investigation ofmetabolic pathways are main topics. The classic approach to study proteomes is to separate proteinsby 2D gel electrophoresis, to excise spots, to cleave the proteinsenzymatically and to investigate the peptides by massspectrometry. However, proteins with extremes in pH andmolecular mass, low abundance proteins and membraneassociated proteins are rarely resolved by 2D PAGE. Moreover, thisprocedure is highly demanding and time-consuming. As an alternative separation technique, we used 2D liquidchromatography prior to mass spectrometric investigation.Ribosomal proteins from Azoarcus-like strain EbN1 served as amodel system of limited complexity (200 proteins). The digestedproteins were separated in two dimensions. One digested non-bacterial protein (Enolase, baker’s yeast) was added to an aliquotof the ribosomal protein mixture, and both samples were analysedby electrospray ion trap analysis. This approach focused on theidentification of the non-bacterial protein and to show aninstrumental setup for promising proceeding analyses.ExperimentalSample preparation: The denitrifying bacterium Azoarcus-likestrain EbN1 (b-Proteobacteria) has been isolated from anoxicfreshwater mud sampled in Bremen, Germany (Rabus and Widdel1995, “Anaerobic degradation of ethylbenzene and otheraromatic hydrocarbons by new denitrifying bacteria”. ArchMicrobiol. 163:96-103). Strain EbN1 was grown in an ascorbate-reduced medium with nitrate (10 mM) and benzoate (4 mM)(Rabus and Widdel 1995). Cells were harvested during theexponential growth phase as described before and stored at 80°C.35302520151050250 5000Probability based mowse scoreNumber of hitsFigure 1: Mascot search result of the 11 protein mixtures. All proteins were identified unambiguously.Proteins with extremes in pH and molecularmass, low abundance proteins and membraneassociated proteins are rarely resolved by 2DPAGE. Moreover, this procedure is highlydemanding and time-consuming.3THEAPPLICATIONSBOOK  September 2003Pharmaceuticalthe valves change their position the trapping column gets in linewith the RP-column. The gradient flushed the peptides from thetrapping column onto the RP-column. The IEX column wasdisconnected from the loading flow, until the next salt injection.RP-chromatographyNanoflow: 200 nl/min, 20-30 µm capillariesColumn: PepMap™ C18, 75 µm, 15 cm (LC-Packings)1-D HPLCNanoflow: 200 nl/min, 20-30 µm capillariesColumn: PepMap™ C18, 75 µm, 15 cm (LC-Packings)Capillary flow: 2.5 µl/min, Column: PS-DVB monolithic columns, 200 µm ID, 5 cm, 60 °C (LC-Packings)ESI MS: esquire3000plusThe ribosomal protein LC-MS/MS data were acquired on anesquire3000plus (Bruker) with a Bruker on-line nanospray source(inner needle tip diameter 10 µm, New Objective; stainless steelunion). HyStar™ 2.3 was used to control 2D chromatography anddata acquisition by esquire 5.1™. The data were processed usingDataAnalysis 3.1™ and BioTools 2.2™. ESI MS: esquireHCTThe in-solution digest LCMSMS data were acquired on anesquireHCT (High Capacity Trap). ResultsComparing complex protein mixtures2D HPLC MS-data were acquired for two samples:1.  ribosomal proteins from EbN12.  ribosomal proteins from EbN1 plus 100 fmol yeast enolase.A software tool which was used to compare the Total IonChromatograms for each salt concentration (both samples)extracted a list of masses which was used as a Preferred Mass Listfor setting up a subsequent LC-MS/MS run (ribosomal proteins plusenolase) in the esquire™ software. The added enolase could beidentified unambiguously. OutlookTo further speed up analysis time, the use of fast monolithic RP-columns can be an option. However, the MS instrument must havea duty cycle fast enough to cope with coeluting peptides showingpeak widths of 3-4 sec. This is given by the esquireHCT with its fastscan speed (ultrascan, 26000 u/sec) allowing MSMS cycle times of0.7 sec (Figure 1). For very sensitive measurements with highresolution and accuracy the slightly slower peptidescan™ (8100u/sec for MS, 26000 u/sec for MSMS) shows better results (Figure2).As 2D-chromatography of  complex mixtures requires a highdynamic range, the improved ion storage capacity of theesquireHCT in combination with an intelligent spectra dependantion charge control (smart-ICC™) appears to be particularlybeneficial.Summary/ConclusionMS-chromatogram traces of two medium complex proteinmixtures were compared. Submitting the resulting masses toMS/MS-fragmentation the additional protein was identifiedunambiguously. Further, an instrumental setup is presented whichpromises a significant progress in the analysis of complex proteinmixtures due to high speed and sensitivity. Bruker Daltonik GmbHFahrenheitstrasse 4,D-28359 Bremen, Germany.tel +49 421 22050, fax +49 421 2205103e-mail: office@bdal.dewebsite: www.bdal.deReader Service 203Time (min)10 15 20 30 35 4025123450Intens. 106Intens. 1050.20.40.60.81.00.0Figure 2: Ultrasensitive Analyses: base peak chromatogramand total ion chromatogram MS(n)-trace of 1 fmol enolase(gradient time 30 min, standard RP-column).

Protein mixtures using two-dimensional chromatography and ion trap MS

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