Multidimensional LC MS for Proteomics Present and Future

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1 Multidimensional LC MS for Proteomics Present and Future Martin Vollmer, Patric Hörth, Cornelia Vad and Edgar Nägele, Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany. Today, two-dimensional on-line or off-line LC MS is state-of-the-art for the identification of proteins from complex proteome samples in many laboratories. Both D LC methods used apply two orthogonal LC separation techniques [e.g., strong cation exchange (SCX) and reversed-phase (RP) separation]. In this article an introduction to the techniques of LC systems for on-line and off-line multidimensional LC and their internal system workflow will be presented. The use of these systems for specific applications in the analysis of proteome samples, depending on their complexity, will be discussed. Additionally an overview of future possibilities in proteomics will be addressed. Introduction The proteome, which can be defined as the complete protein set of a cell derived from a certain time-point and condition of a cell, from a microorganism or from a body fluid is now the focus of many international research groups and pharmaceutical companies. As the sequenced human genome and expression levels of mrna do not describe the current state of a living cell in sufficient detail because of the broad variety of post-translational modifications, scientists discovered the importance of analysing the actual protein composition of a biological system in order to understand complex cellular processes and networks. Proteomics as a scientific discipline emerged with the purpose of rapidly identifying complex protein patterns in a comprehensive manner. 1,, The methodology needed to achieve this ambitious goal must also be powerful enough to detect subtle quantitative and qualitative differences of a protein profile in order to finally identify target proteins and modified variants., Such comprehensive proteome characterization will give new insight to cellular responses for disease pathogenesis such as carcinogenesis, for drug development, as well as ageing, drug effects and environmental damage. In order to identify proteins from a complex mixture of 10 to 10 with a dynamic range of at least 10 it will be crucial to develop technologies with extremely good resolving power on one hand and with extraordinary sensitivity on the other hand. It is obvious that these challenging tasks will not be achieved with a single analytical technology, but with a combination of separation and detection techniques. Present Technical Background D gel electrophoresis: Currently the most frequently used method for separating complex proteomic samples is still D gel electrophoresis (D GE),, because of its high resolving power for proteins. During the D GE procedure the protein mixture is separated by isoelectric focusing in the first dimension and in the second dimension by SDS-PAGE. After in-gel digestion of target proteins, identification is usually achieved by MALDI-TOF- or LC/ESI-QIT mass spectrometry. Despite recent progress of D GE in terms of reproducibility, automation and quantization 9,10,11 D GE still suffers from some major drawbacks. This holds true especially for low-abundance proteins, membrane proteins, proteins with extreme pi values, very large as well as very small proteins, which account for a high percentage of cellular proteins and are often the most promising targets for drug development or disease diagnostics. 1 Multidimensional high performance liquid chromatography (HPLC) methods will fill a gap in the repertoire of tools for proteomics research especially in the analysis of these proteins. 1,1 Nano Flow HPLC Technique The LC vendors active in the field of nano HPLC use different techniques to produce the nano flow with their LC s. Common solutions are passive splitters or syringe s. In contrast to these techniques the nanoflow (Agilent Technologies) is equipped with Electronic Flow Control (EFC) instead of a non-regulated passive splitter, which doesn t allow active control beyond the nanoflow flow-rate. The EFC provides real time adjustment of the flow-rate. The primary flow-rate with the EFC is divided into the nanolitre flow and a waste flow [Figure 1(a)]. To control the active splitting ratio the nanolitre flow is monitored with a nanoflow sensor. This sensor signal drives the split ratio at the electromagnetic proportioning valve (EMPV). The nanoflow sensor 1 consists of a stainless steel capillary, two temperature sensors and a heater around the capillary [Figure 1(b)]. If there is no flow through the capillary the temperature profile around the heater is symmetric. If there is a flow though the capillary the temperature profile shifts downstream. The shift in the temperature profile represents a temperature difference caused by the heat transport of the flowing fluid. This heat transport is proportional to the flowrate. Therefore this sensor measures the flow of the fluid and RECENT APPLICATIONS IN LC MS LC GC Eur., 1(11a) 1 0 (00)

2 delivers a feedback to the EMPV. This ensures flow stability independent from system backpressure fluctuations. The independence of the flow-rate from system backpressure fluctuation is demonstrated when a temporary or permanent partial blockage in the nano sprayer needle in the mass spectrometer ion source occurs (Figure ). During the blockage the pressure raises significantly but the flow-rate is kept constant. Results and Discussion For the separation of complex peptide mixtures the combination of several orthogonal techniques have been used: reversed-phase chromatography (RP) capillary electrophoresis 1,1 size exclusion chromatography (SEC) RP 1 strong cation exchange chromatography (SCX) RP 19,0 affinity chromatography (AC) RP. 1, While the latter approach is mainly applied for more specific functional approaches such as phosphopeptide analysis, SCX RP with the MudPIT technology has, in contrast, been successfully applied for the analysis of comprehensive protein expression profiles from yeast Saccharomyces cervisiae or Plasmodium falciparum., Usually, on-line nano D LC MS is performed using a strong cation exchange (SCX) in series with a reversedphase (RP). In the course of analysis tryptic peptides are gradually eluted in the first dimension by injecting salt solution plugs of increasing ionic strength from the SCX, which is connected to the autosampler and the loading. In the second dimension these peptides are first trapped on a reversed phase (RP) enrichment, which is connected to a micro six-port valve and after a washing step this is switched into the nanoflow path. Finally, the peptides are separated on an analytical RP and analysed by mass spectrometry. 19,0 It has been proven that this methodology, especially in nanoscale, is capable of resolving large proteomes, as well as identifying a subset of proteins expressed under special conditions such as metabolic enzymes or heat-shock proteins., The basic instrument set-up of the nanoflow LC MS system (Agilent Technologies) for twodimensional nano LC MS is shown in Figure (a) and the principle of the two-dimensional LC is shown in Figure (b). The capability to analyse samples containing digested proteins from a gel band or small subproteomes, for example, is given using this method and instrument set-up. However, this method, although frequently used, does not allow an optimization of the injected volume of salt solution to the SCX as the SCX is at a remote distance from its optimum. Thus the non-optimized state of this method is the reason for the distribution of the peptides over more than one fraction, which can dilute them below their detection level or suppress their ionization in nano electrospray by higher abundant peptides in the mass spectrometric analysis. To overcome these limitations an improved method for on-line Figure 1: (a) Principle of active flow splitting with electronic flow control (EFC). (b) Thermal flow sensor measuring principle. From the head EMPV Primary flow Waste flow To the waste (a) To the Flow sensor Column flow (b) T1 T Heater Thermo couples RECENT APPLICATIONS IN LC MS LC GC Eur., 1(11a) 1 0 (00)

3 D LC was developed. 9 In this method the optimized semicontinuous salt solution gradient for the elution of the peptides from the SCX is delivered very precisely with a capillary and the SCX is always maintained under optimum conditions. The eluted peptides are alternately trapped on two enrichment s, which are mounted on a micro 10-port valve and are subjected to reversed-phase separation followed by MS/MS analysis. The basis of this method is illustrated in Figure (c). At the beginning of each RP analysis cycle, the loaded enrichment is exchanged Figure : Robust nanoflow independent from back pressure. A temporary nano sprayer needle blockage in the MS occurs at min. The flow-rate drops and the loss is compensated by the electronic flow control, which leads to an increase in pressure to maintain the original flow-rate. After 0 min the blockage passes the needle and releases the needle tip again. Immediately the flow is kept stable by the electronic flow control. The electronic flow control is even capable of compensating rapid changes in the flow-rate which occur in the system during a valve switch (at min). Norm bar Valve switch 100 bar Temporary needle blockage Pressure Nanoflow: 0 nl min 0.% Time (min) Figure : (a) Nano LC MS system for on-line two-dimensional LC MS. (b) Internal workflow principle of the nano LC system for salt solution plug injection D LC MS. (c) Internal workflow principle of the nano LC system for semi-continuous gradient D LC MS. nd SCX Binary SCX Capillary micro degasser Nanoflow µ WPS 1 st dimension salt gradient in steps 1 st dimension 1 Waste Micro valve and holder nd dimension RP analysis Nanobore cycle Orthogonal nanosprayer and nano Desalt nd dimension Nanoflow LC MSD ion trap XCT Nanobore Waste Nanoflow Ion trap MS Ion trap MS Table 1. Comparison of the highest protein scores for on-line and off-line D LC MS analysis of the yeast cell lysate. Compared with the on-line method, the off-line method yields better resolution which in turn results in greater sequence coverage and, therefore, higher scores in instances where the same proteins are identified by both methods. (Higher scores are emphasized when the difference is > %.) Protein name Peptides Spectra Coverage Score Phosphopyruvate hydratase Phosphoglycerate kinase Pyruvate decarboxylase Glucose kinase Translation elongation factor eef-1 Citrate synthase Pyruvate kinase Hexokinase A Alcohol dehydrogenase 1 Phosphoglycerate mutase 1 Eukaryotic translation initiation factor A- Phosphogluconate dehydrogenase Glycerol--phosphate dehydrogenase Superoxide dismutase RECENT APPLICATIONS IN LC MS LC GC Eur., 1(11a) 1 0 (00)

4 for an empty one by switching the micro 10-port valve. Two different methods, one for SCX chromatography and another one for RP separation are used for chromatographic separation. The semi-continuous salt gradient for the elution of the peptides from the SCX is delivered from a capillary and the gradient for the RP separation is delivered from a nanoflow. The salt gradient is ed in steps; whereas the subsequent steps start with the end concentration of the previous step and ends with the starting concentration of the following step while gradually increasing the salt concentration. Therefore, each step contributes to a semicontinuous salt solution gradient on the SCX, which gives a better resolution of the tryptic peptides and subsequently an increase of 0% in the number of identified proteins compared with the injected salt solution plug method. In comparison to the well-established on-line methodology, off-line D LC MS significantly increases chromatographic resolution of highly complex proteome samples. This improvement is because of the continuous salt solution gradient SCX-chromatography in the first dimension without disrupting RP-chromatography intervals. As a consequence a higher peak capacity and, therefore, a higher number of identified proteins is associated with the off-line D LC MS/MS approach. 0 A micro fraction collection system is used for fraction collection in the microlitre range for the first dimension (SCX). The well plate, containing all collected fractions is then transferred directly to the second dimension the nanoflow RP LC MS system [Figure (a)]. In the second dimension, nano reversed-phase LC MS with the mentioned In comparison to the well-established on-line methodology, off-line D LC MS significantly increases chromatographic resolution of highly complex proteome samples. nanoflow LC MS system is used for the separation of the peptides contained in the eluent obtained from the first dimension. After reinjection the peptides from each fraction are concentrated on a short C1 enrichment located between two ports of the micro valve. The enrichment was then switched into the nanoflow path, the concentrated peptides were eluted, further separated by a gradient with increasing organic solvent on an analytical nanobore and sprayed directly into the MS. The typical system internal workflow for an off-line D LC MS proteome analysis is depicted in Figure (b). This system is capable of analysing large subproteomes as well as whole cell digests of complex eukaryotic cells. For the comparison of the on-line D LC MS methods working with the injected salt solution plugs in increasing concentration or with the ed semi-continuous gradient and the off-line D LC MS method working with a continuous salt solution gradient in the first dimension a whole cell lysate from yeast (Sccacharomyces cerevisiae) was analysed. In general, more proteins with higher score and sequence coverage could be identified with the off-line D LC approach. Table. Distribution of phosphopyruvate hydratase tryptic peptides over the collected fractions in an off-line D LC MS experiment. Sequence m/z MH+ FRAC1- FRAC FRAC FRAC FRAC FRAC FRAC9 FRAC10 FRAC11 FRAC Measure Match score score score score score score score score score 1-1 score (K)AAQDSFAAGWGVMVSHR(S) (K)AVDDFLISLDGTANK(S) (R)GNPTVEVELTTEK(G) (R)IEEELGDNAVFAGENFHHGDKL(-) (K)IGLDCASSEFFK(D) (K)NVNDVIAPAFVK(A) (K)NVPLYKHLADLSK(S) (R)SGETEDTFIADLVVGLR(T) (R)SIVPSGASTGVHEALEMR(D) (K)TAGIQIVADDLTVTNPK(R) (K)VNQIGTLSESIK(A) (K)WLTGPQLADLYHSLMK(R) RECENT APPLICATIONS IN LC MS LC GC Eur., 1(11a) 1 0 (00)

5 Table : Comparison of the on-line D LC methods working with injected salt solution plugs or working with the ed semi-continuous salt solution gradient and the off-line D LC method operated with a real linear continuous salt solution gradient. Method On-line D LC with On-line D LC with ed Off-line D LC with ed injected salt steps semi-continuous salt gradient continuous salt gradient Identified proteins Assigned peptides SCX resolution low high very high Automation high high medium Complexity of set-up medium high medium Inestment low medium high Effort medium medium high Access to SCX fractions no no yes Flexibility low low high The top score proteins are shown in Table 1. To illustrate the full resolution performance of the off-line D LC MS, the distribution of the peptides from the protein phosphopyruvate hydratase over the collected fractions from the first dimension is shown (Table ). This distribution clearly shows that most of the identified tryptic peptides of this protein are only found in one fraction. On account of the better resolution from the SCX used in the first dimension of the off-line D LC approach, it is possible to identify more proteins with more assigned peptides with this methodology. However, each of the described techniques has its own advantages and disadvantages beyond protein identification (Table ). For instance, with an on-line approach the degree of automation is very high and the analysis needs no manual interaction compared with the off-line D LC approach. Therefore the off-line D LC approach enables access to the fractions from the first dimension and allows a chemical or enzymatical modification for example. The higher investment necessary for a D LC system is justified by the improved results obtainable with the off-line D LC method. In particular, the on-line D LC with semi-linear gradient, which gives a better result than the D LC method working with injected salt solution plugs, is highly complex and challenges even an experienced user. Conclusions and Outlook into the Future The various methods of multidimensional LC offer a new complementary tool for proteomics research. This methodology opens up the possibility of analysing proteins, which are not very convenient to handle with the currently used D gel electrophoresis separation approach. The researcher is able to choose the separation method in accordance to the complexity of his proteome sample with the different solutions offered by on-line and off-line D LC. The off-line D LC approach is especially capable of separating even highly complex proteome samples such as whole cell digests. Therefore this may become the method of choice in this field. Understanding of miniaturized systems, such as chip-based systems is continuously developing. Currently investigations on new ways to simplify proteomics tasks to make techniques like LC MS and LC MS/MS more user friendly are under progress. Some include integration of the LC separation Figure : (a) Off-line D LC MS system containing a micro fraction collection system and a nano LC MS system. (b) Internal workflow principle of the off-line D LC MS system. (a) (b) Micro degasser Capillary µ WPS nd 1 st dimension Capillary 1 st dimension: Micro fraction collection system TCC µ port valve µ Fraction collector micro degasser DAD Nanoflow Micro fraction collector µ WPS Transfer of fractions Fractionation with a continuous salt gradient SCX Waste nd dimension: Nano LC/MS system Micro valve and holder Orthogonal nanosprayer and nano Loading Desalt Nanobore MSD ion trap XCT nd Dimension Analysis NanoLC Ion trap MS element on a chip environment and simplification of the connection to the mass spectrometer. The user should benefit from this chip technology its aim is to provide ease of use, robustness and high performance and may open up complicated proteomics applications to the unspecialized LC MS user. RECENT APPLICATIONS IN LC MS LC GC Eur., 1(11a) 1 0 (00)

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