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Chapter 8. Conclusions and perspectives. Summary


Data & Analytics

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Chapter 8 Conclusions and perspectives Summary Chapter 8 Conclusions and perspectives The high efficiency of capillary separations coupled to the high resolving power of modern MS instrumentation, makes
Chapter 8 Conclusions and perspectives Summary Chapter 8 Conclusions and perspectives The high efficiency of capillary separations coupled to the high resolving power of modern MS instrumentation, makes the combination of these two techniques a very powerful analytical tool able to characterise nanogram- to microgram- amounts of complex peptide mixtures. Even though the technology doesn t seem to currently be capable of tackling the full analysis of an entire proteome (either from cellular, tissue or biofluid origin), targeted approaches have demonstrated the power of LC-MS in giving answers to specific questions in molecular and cellular proteomics. In fact, the characterisation of separate cellular compartments or of specific classes of proteins (phosphorylated proteins, membrane proteins, low molecular weight proteins) has proved to be very successful, giving tremendous insights in the protein biochemistry field. A very promising on-going research in protein/peptide profiling by MS or LC-MS is disease biomarker discovery. This exploration is still in its infancy, even though first reports in the field date back already to 3-4 years ago. Perspectives in proteomics biomarker discovery are bright: new MS-based technologies have only recently entered many clinical settings, and they will certainly play a major role in new achievements and discoveries during the next few years. The clinic is now realising potential and limitations of these new proteomics technologies. The utopia of comprehensive proteome analysis, especially in the case of plasma/serum, stays for the moment in a far horizon. Instead, recent undertakings in the biofluid proteomics arena rely on more targeted approaches, or are fully aware of the limitations of current technology and its inability of screening in high throughput thousands of gene products spanning 9 orders of magnitude in concentration. Optimal choices of sample sets and sample pretreatment steps will be strategic in the design of successful studies. A role of paramount importance will also be played by the development of data analysis tools able to extract knowledge out of the ever-increasing level of information produced at the analytical level. Today s LC-MS technology and competing technologies like 2DE allow for parallel protein determination and accurate quantitation as it was not possible in the past. This parallel screening of hundreds of proteins could possibly lead to the discovery of multiple markers of disease, something which surpasses the current, limited concept of one-disease, one-marker, often inadequate to characterise diseased status in its complexity. The greatest pitfall of the most powerful analytical technologies available today (2D LC-MS, 2DE coupled to fractionation techniques) is their inherent low throughput. 169 Conclusions and perspectives - Summary Thus, biomarker discovery studies undertaken with state-of-the art technologies can, unfortunately, be slow in delivering their exciting new findings. Future advances in LC-MS proteomics from the technology side will have to be directed towards the most important drawbacks of today s technology. The issue of proteome coverage and dynamic range is still an open problem in proteomics. Dynamic range of detection of most mass spectrometers is not sufficient to tackle global proteomic investigations. Thus, improvements in this field will be of special interest. From the qualitative analysis side, tandem MS on intact proteins, the so-called top-down approach to MS proteomics, has been described recently. Performing MS analyses directly at the protein level, without tryptic digestion, would have advantages in terms of keeping information about protein isoforms and making analysis protocols simpler. The coming years will tell if top-down proteomics will prove sufficiently robust and universal to challenge bottom-up, shotgun approaches. A key to success will probably consist in finding an effective intact protein separation technique able to be coupled on-line to MS (e.g. capillary electrophoresis or LC in monolithic columns), in order to add an extra, orthogonal dimension to MS separation. In fact, intact protein separations are still usually performed in conditions not compatible with direct MS introduction. While proteome coverage, throughput and data analysis are still the biggest issues in comprehensive LC-MS based proteomics, sensitivity is another very important analytical parameter which still is the focus of many research efforts. The concentration-dependent-like nature of electrospray is already pushing the scale of analytical separations to flow rates in the mid-nanoliter/min range, allowing subnanograms amounts of proteins to be characterised at least at the qualitative level. Further advancements in miniaturisation of analytical separations (nanolc, CE, CEC) coupled to mass spectrometry will provide analytical tool capable of looking, with the specificity of mass spectrometry, at extremely low levels of proteins or metabolites in biological samples. Discovery of new protein-protein interactions or proteome/metabolome characterisation of very limited cell populations (down to single cell level) will be two exciting fields where new improvements in separation/mass spectrometry miniaturisation will find successful application. 170 Chapter 8 Summary Sensitive and robust MS-based analytical technology along with effective and fast analysis of data produced, are two major needs in current research on protein and peptide analysis, which aims at characterising an increasingly large portion of entire cellular, biofluid or tissue proteomes. Because most situations in life science research are sample limited, miniaturisation of analytical separations is an active field of research, especially in the case the chosen detector has apparent concentration-dependent behaviour, like in the case of ESI. Chapter 3 described approaches to the coupling of low-flow, high sensitivity CEC separations to mass spectrometry with either a nanoes interface or a sheath flow, microspray interface. Separation speed and improved ruggedness of the CEC-MS system supported by the sheath flow liquid interface was obtained at the cost of a drop in sensitivity of about fold. In Chapter 4, high sensitivity separations were obtained by in-house fabricated nanolc columns. The packing procedure described allowed fabrication of extremely narrow-bore capillaries, down to 30 μm ID, delivering sensitivity for peptide detection at least in the low-femtomolar range. Improvement of analytical tools can bring real advancements only if the increasing size of the information produced can be efficiently managed by specially developed informatics tools. The full exploitation of LC-MS data by informatic analysis is an ongoing research goal, which is continuously challenged by the increasing resolution power of mass spectrometers. Data analysis on hyphenated capillary separations of increasing complexity have been described in Chapters 5, 6 and 7. An early demonstration of PCA analysis on LC-MS peptide profiles has been presented in Chapter 5. LC-MS data on digests of a standard protein (serum albumin) obtained from three different manufacturers could be clustered in a PCA plot. Factor spectrum analysis could point at significant peaks responsible for the PCA separation. Chapter 6 has described a successful approach to the characterisation of an enzymatic protein modification (acetylation). Tryptic digests of the modified protein and a control sample were subjected to MALDI-TOF analysis and subsequently to LC-MS/MS analysis of selected peptides. MALDI-TOF analysis was used first so that simple, superimposable MS spectra from unknown and control samples could be acquired. By overlaying the two simple spectra it was possible to easily discover new peaks, potentially deriving from the expected protein modification. In a second phase, targeted LC-MS/MS analysis on the peaks of interest was performed. Seven acetylated peptides were identified in the study. As an alternative approach, new informatics tools 171 Conclusions and perspectives - Summary for direct comparison of LC-MS data were used, which allowed to by-pass the initial MALDI-TOF profile step. Direct LC-MS analysis of the two protein digests under consideration was undertaken, using automated peak picking and principal component analysis tools described in Chapter 5. Four of the seven acetylated peptides were successfully found by this alternative, operator-free approach. Finally, automated data analysis was applied to 2D LC-MS data obtained using an original optimised method for off-line 2D LC-MS capable of long-lasting unattended operation. A complex proteomic sample of secreted proteins from U937 human monocytic cell line was analysed. Optimised informatic tools for peak picking and alignment were used to process the 2D LC-MS information and clustering of the data in PCA and PCDA plots revealed significant differences between proteomes obtained in different stress conditions. As opposed to the direct LC-MS/MS approach aiming at identifying as many proteins as possible in data-dependent acquisition mode, this approach aimed at quantitatively characterise any peak present in the data, and subsequently achieve targeted protein identification only on peptides showing major up- or down-regulation between the different clustered groups. 172 Chapter 8 Samenvatting Gevoeligheid en robuustheid, gecombineerd met effectieve en snelle analyse van de geproduceerde data, zijn op dit moment de belangrijkste elementen voor de masspectrometrische analyse van zo veel mogelijk eiwitten (proteomics) en peptiden (peptidomics) in biologische materialen zoals cellen, lichaamsvloeistoffen of weefsels. Veelal is in het life sciences onderzoek de beschikbare hoeveelheid monster een beperkende factor. Bij gebruik van concentratiegevoelige detectiemethoden, zoals electrospray (ES) massaspectrometrie (ESI-MS) is miniaturisering van de analytische scheidingtechniek een interessante manier om de gevoeligheid van de methode te verbeteren. Hoofdstuk 3 beschrijft de koppeling van een geminiaturiseerde scheidingstechniek, CEC, met MS via een nano ES interface en een sheath flow microspray interface. Deze laatste aanpak resulteerde in een kortere analysetijd en betere robuustheid van de CE-MS methode, echter dit ging gepaard met een verlies in gevoeligheid van ongeveer een factor 20 tot 40 ten opzichte van de nano ES interface. In Hoofdstuk 4 wordt beschreven hoe de gevoeligheid sterk verbeterd kan worden met zelfgemaakte nanolc kolommen. De beschreven procedure maakt het mogelijk om capillaire nanolc kolommen te fabriceren met een bijzonder kleine inwendige diameter van 30m. Dit resulteerde in een sterk verbeterde gevoeligheid voor de analyse van peptiden,met concentraties in het lage femtomolair gebied. Verbetering van analytische technieken moet als doel hebben het genereren van meer data van betere kwaliteit om daarmee gedetailleerde en kwantitieve informatie van zo veel mogelijk eiwitten in biologische materialen te verkrijgen. Echte vooruitgang wordt alleen geboekt als de toegenomen hoeveelheid complexe data efficient wordt verwerkt tot relevante informatie. Dit is gerealiseered met speciaal ontworpen dataverwerkings-software. De volledige exploitatie van LC-MS data met dataverwerkings-software is een steeds belangrijker wordend onderzoeksgebied. Automatische verwerking en analyse van complexe LC-MS data is beschreven in hoofdstuk 5, 6 en 7. De toepassing van multivariate analyse (PCA) op LC-MS peptide profielen, verkegen na enzymatische digestie van het eiwit albumine afkomstig van 3 fabrikanten/leveranciers is beschreven in hoofdstuk 5. De PCA score plot toont 3 clusters, wat duidt op verschillen tussen de 3 verschillen albumine referenties. De bijbehorende factor spectra laten zien welke pieken in de data verantwoordelijk zijn voor deze verschillen. Hoofdstuk 6 beschrijft een vergelijkbare benadering voor de karakterisering van enzymatische eiwitmodificaties (acetylering). Na enzymatische digestie van het 173 Conclusions and perspectives - Summary gemodificeerde eiwit en het oorspronkelijke eiwit zijn peptiden gemeten met MALDI- TOF MS en LC-MS/MS. Mogelijke enzymatische eiwitmodificaties zijn eenvoudig gevonden door de twee MALDI spectra over elkaar te leggen en naar verschilpieken te zoeken. In een tweede fase, is LC-MS/MS analyse van de gevonden verschilpieken uitgevoerd. Zeven geacetyleerde peptiden zijn geidentificeerd met deze methode. Ter vergelijking is de methode uit hoofdstuk 6 hier ook gebruikt, d.w.z. de directe LC-MS analyse van de 2 gedigesteerde eiwitten gevolgd door automatische dataverwerking (peak picking) en PCA. Met deze geautomatiseerde werkwijze werden vier van de zeven geacetyleerde peptiden met succes gevonden. Hoofstuk 7 beschrijft een echte proteomics toepassing van deze werkwijze. Automatische dataverwerking en analyse werd toegepast op LC-MS data verkregen door de analyse van door U937 cellen (humane monocyten cellijn) uitgescheiden eiwitten met een geoptimaliseerde methode voor off-line 2D LC-MS. Het doel was het vinden van veranderingen in de samenstelling van de uitgescheiden eiwitten als gevolg van verschillende behandelingen van de cellen. PCA en PCDA analyse resulteerde in het vinden van een groot aantal significante verschillen. Deze werkwijze is fundamenteel anders dan de standaardmethode gebaseerd op LC-MS/MS identificatie van zoveel mogelijk eiwitten met data dependent MS/MS. De hier beschreven aanpak richt zich primair op de volledige kwantitatieve karakterisering van alle peptiden. Alleen de peptiden die significant verschillen in concentratie tussen de monsters worden met MS/MS geidenticeerd, en daarmee ook de eiwitten waarvan ze afkomstig zijn. 174
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