SIMS2015 Session LS1-WeM: Life Science and Biotechnology

Wednesday, September 16, 2015 8:40 AM in Grand Ballroom I
Wednesday Morning

Time Period WeM Sessions | Abstract Timeline | Topic LS Sessions | Time Periods | Topics | SIMS2015 Schedule

Start Invited? Item
8:40 AM Invited LS1-WeM-1 Imaging of Molecular Chemistry in Biological Specimens by Next-Generation TOF-SIMS
Gregory L. Fisher (Physical Electronics); Nina Ogrinc Potocnik, Anne L. Bruinen, Bryn Flinders (Maastricht University, Netherlands); Takuya Miyayama (ULVACPHI, Japan); Shin-ichi Iida (ULVAC-PHI, Japan); John Hammond, Scott Bryan (Physical Electronics); Ron M.A. Heeren (Maastricht University, Netherlands)
We employ a new and patented [1] imaging MS/MS (tandem imaging mass spectrometer), exploiting the unique characteristics of the TRIFT spectrometer of the PHI nanoTOF II (Physical Electronics, USA) TOF-SIMS instrument, for the unambiguous identification of molecular chemistry in biological specimens. Tandem mass spectrometry is based on the selection of a high mass molecular ion (precursor ion) from the precursor ion spectrum (MS1), followed by energetic activation and fragmentation of the precursor ion and, finally, collection of the fragment ion (product ion) spectrum (MS2). The use of MALDI MS/MS, for example, is reviewed by McDonnell and colleagues [2].

The use of MALDI imaging MS/MS has several inherent limitations. The large sampling volume consumes the sample in a single imaging analysis. The typical lateral resolution is > 30 um. An ionization matrix, which perturbs the intrinsic molecular chemistry and which commonly degrades the lateral resolution, must be applied. Lastly, it has not been possible with present instrument designs to simultaneously collect the precursor ion (MS1) and the product ion (MS2) spectra, much less the imaging MS data sets.

TOF-SIMS, by comparison, has a sampling depth of only ~ 2 nm and routinely achieves a lateral resolution of < 300 nm at high mass resolution. An ionization matrix is not employed in TOF-SIMS analysis so only the intrinsic molecular chemistry is probed. The TRIFT mass spectrometer produces inherently low spectral background due to metastable post-source decay (PSD) product ion filtering. Finally, and perhaps most important, the ion optic design of the TRIFT mass spectrometer allows simultaneous collection of the precursor ion (MS1) and the product ion (MS2) imaging MS data sets. That is to say, our new approach enables simultaneous surface screening with MS1 imaging and targeted identification with MS2 imaging.

We have brought this new analytical capability to bear in two studies: one concerns the chronological ingestion of drugs of abuse as examined in human hair specimens, and the other involves the differentiation and identification of intact lipids in spleen specimens infected with F. novicida. In each study, the ability to collect imaging MS/MS (tandem IMS) data was vital in addressing the theory or claim. All tandem IMS analyses were conducted using a PHI nanoTOF II which was equipped with both a Bi cluster liquid metal ion beam and a C60 cluster ion beam.

[1] P.E. Larsen, J.S. Hammond, R.M.A. Heeren and G.L. Fisher, Method and Apparatus to Provide Parallel Acquisition of MS/MS Data, U.S. Patent 20150090874, 02 April 2015.

[2] L.A. McDonnell and R.M.A. Heeren, Mass Spectrom. Rev. 26 (2007) 606-643.

9:20 AM LS1-WeM-3 ToF-SIMS using a GCIB for Studies on Lipid Distribution following Myocardial Infarction
Sanna Sämfors (Chalmers University of Technology, Sweden); Maria Heyden, Jan Borén (University of Gothenburg, Sweden); Andrew G. Ewing, John S. Fletcher (Chalmers University of Technology, Sweden)

Cardiovascular disease is the leading cause of death worldwide. Understanding the mechanism of breakdown and repair of cardiac tissue following myocardial infarction will help in development of better treatment methods.

It has been shown that lipids accumulate in different areas of the heart following infarction. The amount of lipids accumulating can be connected to how well the heart recovers after an infarction. In this project we have studied the spatial distribution of these accumulated lipids with the help of ToF-SIMS. ToF-SIMS is a label free analytical technique, which allows us to study the localization of unknown compounds without the need for chemical or biological labeling.

In our study a J105 ToF-SIMS instrument equipped with a 40 Kv argon cluster ion gun was used to determine the spatial distribution and composition of lipids in mouse cardio tissue after surgically induces infarction. 10 μm thick slices were analyzed either frozen hydrated or freeze-dried in both positive and negative ion mode.

Results show that differences in intensity of the m/z peaks connected to different lipids can be detected between the hypoxic region of the heart and normal tissue region as well as specific accumulation of species at the boundary of the damaged region. Different spatial distributions of lipids were detected in both positive and negative ion mode providing insight into the changes in lipid metabolism following infarction.

9:40 AM LS1-WeM-4 ToF-SIMS Analysis of Amyloid Beta Aggregation forms on Different Lipid Membranes
Yuta Yokoyama (Seikei University, Japan); Toshinori Shimanouchi (Okayama University, Japan); Hideo Iwai (NIMS, Japan); Satoka Aoyagi (Seikei University, Japan)

Amyloid beta (Aβ) peptides are considered to have a strong connection with Alzheimer’s disease. Aβ rarely aggregates on soft lipid membranes of the liquid crystal phase, while it forms α-helix or β-sheet on hard lipid membranes of the gel phase. The β-sheet Aβ peptides would aggregate and form fiber structures and then have toxicity to cells. In the previous study [1], Aβ-adsorbed three lipids were evaluated using time-of-flight secondary ion mass spectrometry (ToF-SIMS), and the aggregation differences depending on the lipid membrane condition were indicated by ToF-SIMS imaging. Aβ effectively adsorbs on model membranes including a lipid and carbohydrate-conjugated lipid such as ganglioside. It is expected that cholesteryl-β-D-glucoside (β-CG) similar to ganglioside.

ToF-SIMS is one of the most effective tools in imaging of biological materials because it has extremely high surface sensitivity for detecting less than one molecular layer (less than 2 nm) and high spatial resolution (less than 100 nm). In general, ToF-SIMS spectra contain useful information including molecular ions and many fragment ions related to a variety of co-existing materials, which makes the interpretation of ToF-SIMS spectra difficult. Therefore, data analysis methods such as principal component analysis (PCA) [2, 3] and G-SIMS [3, 4] have been employed in the analysis of ToF-SIMS data.

In this study, the model samples, Aβ(1-40) on the mixed membranes of different ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and β-CG were measured with ToF-SIMS (TRIFT V nanoTOF, ULVAC-PHI, Inc., Chigasaki) using 30 Kv Bi+ and Bi3+ primary ion beams. The ToF-SIMS data was analyzed by using the data analysis methods, PCA, multivariate curve resolution (MCR), G-SIMS and g-ogram, for extracting important fragment ions related to target molecular ions.

As a result, the aggregation difference of Aβ(1-40) depending on the ratio of DMPC and β-CG in the lipid membranes was indicated from PCA results. It is suggested that these data analysis methods are effective in the analysis of complicated ToF-SIMS data including imaging of bio-molecules. The findings from this study are important for a further study for investigating the aggregation mechanism of Aβ.

References

[1] S. Aoyagi, T. Shimanouchi, T. Kawashima, H. Iwai, Anal. Bioanal. Chem., 407, 2859-2863 (2015).

[2] M. S. Wagner, D. G. Castner, Langmuir, 17, 4649-4660 (2001).

[3] I. S. Gilmore, M. P. Seah, Appl. Surf. Sci., 161, 465-480 (2000)

[4] R. Ogaki, I. S. Gilmore, M. R. Alexander, F. M. Green, M. C. Davies, J. L. S. Lee, Anal. Chem., 83, 3627-3631 (2011).

10:20 AM LS1-WeM-6 The Influence of Primary Ion Beam Energy on Peptide Fragmentation by Means of ToF-SIMS Using Huge Cluster Ion Sources
Satoka Aoyagi, Yuta Yokoyama (Seikei University, Japan); Makiko Fujii (Kyoto University); Jiro Matsuo (Kyoto University, Japan); John S. Fletcher (Chalmers University of Technology, Sweden); Nicholas Lockyer, John Vickerman (University of Manchester)

It is crucial to obtain chemical mapping of biomolecules for evaluating biological tissues, biomaterials and bio-devices. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using huge cluster ion beams such as C60 and Ar cluster ions is one of the most promising techniques for biological and medical applications. However, it is still difficult to detect strong enough molecular ions of macromolecules such as proteins and peptides for imaging. Although fragment ions that are detected more abundantly than molecular ions would be useful for imaging, it is often difficult to determine their origins due to peak overlap and the influence of co-existing materials. Therefore, it is important to interpret fragment ions from macromolecules for the evaluation of biomolecules and the imaging of biomaterials and tissues.

It has been reported that ToF-SIMS with Ar clusters and C60 ion beams provides series of peptide fragment ions from peptides that are similar to collision-induced dissociation (CID) spectra [1]. The support of data analysis is still necessary for interpreting ToF-SIMS data containing unknown peptides because ToF-SIMS spectra are more complicated than conventional CID mass spectra. In this study, several peptides having different molecular weights from approximately 500 to 1600 u were measured with huge cluster ion beams of different energies per atom in order to distinguish fragment ions related to a target molecule from those related to the others.

As a result, there are criteria in terms of primary ion energy for generating typical peptide fragment ions such as a, b and y-type ions that are usually detected by CID. For instance, the typical peptide fragment ions larger than 400 u are strongly detected by using a primary ion beam less than several 10 Ev/atom. On the other hand, the peptide-related ion generation is suppressed under extremely low energy of less than approximately 3 Ev/atom. Thus, it is suggested that the consideration of energy-dependent fragmentation is helpful for interpreting peptide spectra.

[1] S. Aoyagi, J. S. Fletcher, S. Sheraz, T. Kawashima, I. Berrueta Razo, A. Henderson, N. P. Lockyer, and J. C. Vickerman, Anal. Bioanalytical Chem. (2013) 405, 6621-6628.

10:40 AM LS1-WeM-7 Studying Platelet Activation by Time-of-Flight Secondary Ion Mass Spectrometry with Principle Component Analysis
Ilya Reviakine, Alessia Donati (Karlsruhe Institute of Technology, Germany); Swati Gupta (CIC biomaGUNE, Spain)

Regulation of platelet activity holds the key to the delicate balance between haemostasis and thrombosis. On the one hand, these anuclear cell fragments circulating in blood catalyze clot formation at the site of injury—a mechanism for limiting traumatic blood loss. On the other hand, they are well-known for their sinister role in cardiovascular disorders (CVDs), the number one cause of death world-wide. They are also involved in a diverse variety of processes: wound healing, angiogenesis, maintenance of vascular wall integrity, pathogen recognition, and the development of cancer metastases. Understanding the mechanisms underlying the regulation of this diverse set of platelet functions remains challenging. Part of the challenge arises from the limited number of (immuno)labeling strategies that could be used to distinguish between differently activated platelets. Therefore, the focus of our group has become on developing new labels and label-free methods for studying platelet functional diversity. Here, we present the first results of ToF-SIMS/PCA analysis of fixed platelets, from different donors, activated with different agonist, or adsorbed on model biomaterial surfaces. Relatively small size of the platelets (~ 4 um in the resting state) represents a particular challenge. Application of different instrument operating modes to address this challenge will be discussed. We demonstrate that it is possible to distinguish between platelets activated in different ways, paving the way for further studies.

11:00 AM LS1-WeM-8 Biomolecular Investigation of Neurodegenerative Diseases and Brain Plasticity with TOF SIMS Tandem Imaging MS
Nina Ogrinc Potočnik (Maastricht University, Netherlands); Gregory L. Fisher (Physical Electronics); Jelle Praet, Julie Hamaide (University of Antwerp, Belgium); Anne L. Bruinen (Maastricht University, Netherlands); Annemie van der Linden (University of Antwerp, Belgium); Ron M.A. Heeren (Maastricht University, Netherlands)

Lipids and small metabolites play an important role in studying early stages and progression of different neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and dementia, as well as plasticity during brain development [1, 2]. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has proven to be a useful technique for localizing specific biomarkers from the tissue surface but lacks proper means for correctly assigning their structural properties. An innovative tandem SIMS method with augmented dutycycle is employed as a unique visualization tool, capable of identifying and characterizing molecules directly from the tissue surface with sub-micrometer resolution.

A PHI nanoTOF II (Physical Electronics, USA) is equipped with an in beam angled tandem TOF for MS2 analysis [3]. In the MS2 analysis the desired precursor ion from the MS1 spectrum undergoes subsequent fragmentation by collision induced dissociation (CID) and the resulting fragments are detected by the second detector. The major advantage of the developed system is simultaneous MS1 and MS2 data collection and relentless imaging of the same region below the static limit.

The capabilities of the TOF-SIMS tandem imaging MS technique are presented in two different case studies. In the first study we determined the lipid and lipid metabolite presence and as well as their distributions in brain tissue sections of healthy wild type and transgenic APPKM670/671NL/PS1L166P mice [4]. The targeted lipids and their metabolites are potential biomarkers for amyloid plaque formation which is a major hallmark in Alzheimer’s disease. In the second example we show the fatty acid and lipid distributions which are important contributors in signalling pathways of song nuclei [5] and brain plasticity during the development stages in songbirds, i.e. Zebra finch (Taeniopygia guttata). These studies, carried out on advanced instrumentation, can rapidly deepen our understanding of biological processes during neurodegenerative disease progression and brain development.

[1]M. Stoeckli, R. Knochenmuss, G. McCombie, D. Mueller, T. Rohner, D. Staab, and K.-H. Wiederhold, Meth. Enzymol., vol. 412, pp. 94–106, 2006.

[2] M. Shariatgorji, A. Nilsson, R. J. A. Goodwin, P. Källback, N. Schintu, X. Zhang, A. R. Crossman, E. Bezard, P. Svenningsson, and P. E. Andren, Neuron, vol. 84, no. 4, pp. 697–707, 2014.

[3] P.E. Larson, J.S. Hammond, R.M.A. Heeren and G.L. Fisher, Method and Apparatus to Provide Parallel Acquisition of MS/MS Data, U.S. Patent 20150090874, April 2015.

[4] R. Radde et al. EMBO, vol. 7, pp.940–946, 2006.

[5] K. R. Amaya, J. V. Sweedler, D. F. Clayton, J. Neurochem. Vol. 118, 499–511, 2011.

11:20 AM LS1-WeM-9 Identification of Biomarkers for Human Inflamed and Dysplastic Colon Tissues by Time-Of-Flight Secondary Ion Mass Spectrometry and Multivariate Analysis
Marco Urbini (University of Rome "Roma Tre", Italy); Valentina Petito, Franco Scaldaferri, Antonio Gasbarrini (Catholic University of the Sacred Heart, Policlinico "A. Gemelli", Italy); Francesco de Notaristefani, Luca Tortora (University of Rome "Roma Tre", Italy)

Ulcerative colitis (UC) is a chronic inflammatory condition of the colonic mucosa starting from the very superficial layer and limited to lamina propria region, associated also to chronic inflammation induced colonic cancer. Colorectal cancer is one of the most prevalent and fatal cancers that causes a relevant number of mortalities worldwide. As with many other cancer types also in this case a small number of markers have been identified. However, little have been reported in literature regarding the direct comparison between the inflamed and precancerous colon tissues. In fact, to prevent the development of tumour, the chemical differences between normal, inflamed and precancerous (dysplasia) colon tissues play an important role on the knowledge of colon cancer progression. Aim of our work has been to detect new molecular targets in Ulcerative Colitis and dysplastic diseases.

Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is an highly sensitive surface characterization MS technique that offers a new way of studying biomarkers at the microscopic level in biological materials. ToF-SIMS, after the advent of cluster ion sources, has allowed the detection of several high molecular weight fragments with high mass resolution and sub-micrometer spatial resolution such as amino acids and lipids, providing elemental and molecular surface chemical information.

We have obtained intestinal mucosal samples from healthy, inflamed and dysplastic area in course of diagnostic endoscopy. Biopsies have been snap frozen immediately and then fixed for cryosectioning before freeze-drying. The ToF-SIMS analysis have been performed using cluster primary ion source of Bismuth (Bi3++). After measurements, we have applied multivariate analysis, in particular principal component analysis (PCA) and hierarchical analysis, in order to reduce the size of large data sets and grouping samples in cluster with minimal loss of information.

Mass spectra analyses along with PCA revealed amino acids and lipids differences. In particular, through PCA on amino acids fragments, glutamic acids (58 m/z) have been associated with inflamed tissue whereas fragment at 91 m/z (tyrosine) presented greater abundance in dysplastic tissues. Furthermore, positive mass spectra show in normal tissue the presence of MAG, DAG, TAG and cholesterol whereas in inflamed and dysplastic tissues the only presence of the cholesterol fragments at 369 m/z and 385 m/z.

In conclusion, ToF-SIMS plus multivariate analyses represent a tool to investigate and diagnose human colon disease such as inflammatory bowel disease and cancer, giving information about biomarkers responsible for the chemical differences.
11:40 AM LS1-WeM-10 Identification of Tattoo Pigments and Tattoo Pigment Fragments after Laser Treatment
Harald Jungnickel (German Federal Institute for Risk Assessment, Germany); H.P. Berlien (Elisabeth Clinic, Berlin, Germany); Peter Laux, Ines Schreiver, Andreas Luch (German Federal Institute for Risk Assessment, Germany)

Tattooing exists already since the Neolithic period. With the invention of the electric tattooing machine tattoos started to conquer the world. In Europe and the US tattooing becomes more and more a popular mainstream accessory, comparable to piercings or jewelry. Astonishingly little is known about human health risks arising from long time effects of tattoos during pigment aging or about possible health risks of tattoo pigment fragments resulting from laser treatments used for tattoo removal. Valid data on tattoo pigments and their fragments resulting from laser removal within skin samples together with information about possible degradation metabolites occurring over time are key pre-requisites in a valid health-based risk assessment of tattooing. Imaging Mass Spectrometry (ToF-SIMS) in combination with multivariate statistical data analysis was used to assess possible fragmentation patterns of tattoo inks after laser treatment. For the first time a multivariate statistical model was developed to discriminate between untreated tattoo color samples and tattoo color fragment samples after laser treatment. The system could be used to assign unknown samples, tattoo pigments or tattoo laser fragment samples into their respective group. Ultimately the method could be used for the development of a health based risk assessment of tattoo pigment fragments and their distribution within skin samples after laser removal but could also be used to design laser tattoo removal parameters and removal laser algorithms for tattoo pigments, which do not produce unwanted laser fragments that might cause adverse health effects.

Time Period WeM Sessions | Abstract Timeline | Topic LS Sessions | Time Periods | Topics | SIMS2015 Schedule