Journal of Archaeological Science

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Journal of Archaeological Science"

Transcription

1 Journal of Archaeological Science 36 (2009) Contents lists available at ScienceDirect Journal of Archaeological Science journal homepage: Extraction and sequencing of human and Neanderthal mature enamel proteins using MALDI-TOF/TOF MS Christina M. Nielsen-Marsh a, Christin Stegemann b, Ralf Hoffmann b, Tanya Smith a,c, Robin Feeney a, Michel Toussaint d, Katerina Harvati a, Eleni Panagopoulou e, Jean-Jacques Hublin a, Michael P. Richards a,f, * a Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, Germany b Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, Leipzig, Germany c Department of Anthropology, Harvard University, 11 Divinity Avenue, Cambridge, MA 02138, USA d Direction de l Archéologie, Ministère de la région Wallone, 1 Rue des Brigades d Irlande, 5100 Namur, Belgium e Ephoreia of Palaeoanthropology-Speleology of Southern Greece, Ardittou 34b, Athens, Greece f Department of Anthropology, University of British Columbia, Vancouver BC, Canada V6T 1Z1 article info abstract Article history: Received 22 December 2008 Received in revised form 29 March 2009 Accepted 3 April 2009 Keywords: Proteomics Enamel Neanderthal MALDI-TOF-TOF We report here the first results of a method to extract and sequence mature enamel proteins from modern human and Neanderthal tooth enamel. Using MALDI-TOF/TOF mass spectrometry and a combination of direct sequencing and peptide mass mapping we have sequenced a peptide from the tyrosine-rich amelogenin peptide (TRAP) of the X isoform of the amelogenin protein for modern and recent human samples. We also report our results from two Neanderthal enamel samples where we were also able to recover fragments of the TRAP protein, which had a similar sequence to the modern human samples. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Proteins and DNA, when successfully extracted from ancient skeletal remains, can be used to provide information on the climate, genetics, diet and ecology of ancient humans and Neanderthals and associated animals (Lee-Thorp and Sponheimer, 2006; Nielsen- Marsh et al., 2005; Lalueza-Fox et al., 2006). However, despite some notable successes, neither collagen, the most abundant protein in bone, nor DNA, survive well in the burial environment and studies relying on these molecules to provide usable biomolecular information are frequently limited by both time and location. Though the relatively recent successes with osteocalcin offer another informative molecule for archaeological scientists to utilise (Nielsen-Marsh et al., 2005; Ostrom et al., 2006), the survival potential of all these molecules (DNA, collagen and osteocalcin), is limited by the bone substrate in which they are embedded. To date, * Corresponding author. Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, Leipzig, Germany. Tel.: þ ; fax: þ address: (M.P. Richards). only a few early modern human and Neanderthal remains have contained enough surviving biomolecules to provide information on the life histories of these individuals; and these two species are the only two members of our genus for which any biomolecular information has been successfully recovered. If we are to increase our chances at obtaining useable organic material from valuable, but poorly preserved specimens, or perhaps more significantly, investigate deeper into our lineage and study older hominin fossils, from more hostile environments, then more enduring molecules need to be identified and utilised. Fossil tooth enamel represents an, as yet, untapped source of biomolecular information which may allow us to extend palaeodietary and phylogenetic studies back to our earlier ancestors. Enamel is the hardest substance in the body and is, typically, the best-preserved element in ancient skeletal remains. A variety of information have been obtained from fossil teeth, both at the physical (Dean, 2006; Smith et al., 2007) and also chemical levels, where analyses on fossil tooth enamel have been employed to provide palaeoclimatic and palaeodietary information (Nielsen- Marsh et al., 2005; Koch, 1998; Passey and Cerling, 2006). However, although enamel mineral has been used to provide life history data for fossil hominins, as yet, no such valuable information has ever /$ see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi: /j.jas

2 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) been obtained from biomolecules extracted from fossil enamel extracts. It is the heavily mineralised structure of enamel that gives its hardness and durability, leading to survival over very long time periods, but it is also this characteristic that may potentially hinder our ability to extract useable quantities of biomolecules for molecular palaeontological research. Developing enamel contains around 30% proteinaceous material, but approximately 90% of all protein is lost during maturation (Glimcher et al., 1977). Mature enamel contains only traces of structural proteins (around 0.1 w 0.03%), including enamelin and the sheath protein ameloblastin (Glimcher et al., 1990; Fincham et al., 1999). Also present, in low but isolatable quantities, is a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP (Brookes et al., 1995). Amelogenin makes up 90% of the protein content of immature enamel; however as enamel mineralization progresses, amelogenin is subjected to proteolytic activity and eliminated from the enamel environment, leaving only low molecular weight peptides. TRAP is one hydrolytic product of amelogenin, cleaved during maturation between Trp45 and Leu46 by the enzyme EMSP1; also formed is the leucine-rich amelogenin peptide, or LRAP. The amelogenin gene is expressed from both the X (AMELX) and Y (AMELY) chromosomes resulting in a difference between the amelogenin protein sequences (Salido et al., 1992). The first 28 amino acids at the N-terminus are identical. The methionine at residue 29 of the Y form is conserved, but absent from the X form, due to a 3-bp deletion in the AMELX gene. As there are only very small quantities of protein present in mature enamel, and there is likely to be even less in degraded fossil specimens, we used matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) in this study to identify and sequence peptides and proteins recovered from the enamel extracts. MALDI-TOF/TOF MS has been successfully used in previous studies for sequencing protein extracts from ancient bones (Nielsen-Marsh et al., 2005, 2002; Ostrom et al., 2006). A combination of high sensitivity enabling femtomolar quantities of proteins and peptides (which do not need to be intact) to be directly sequenced, and a tolerance for low quantities of salts make MALDI-TOF/TOF MS an ideal tool when working with fossil extracts, which often require intensive, multiple purification steps. 2. Results Following tryptic digestions, peaks were observed in all samples and in some cases were sufficiently high for sequencing after highenergy collision of selected ions. Peptide mass fingerprint data were submitted to FindPept (SwissProt; and fragment ion data were submitted to the Mascot MS/MS Ion Search tool ( to obtain identification of peak masses. The FindPept search tool enables the identification of peptides that result from unspecific cleavage of proteins from their experimental masses in addition to identifying masses that correspond to autolysis and keratin digestion. Keratin is a common contaminant in MALDI spectra and can be a serious issue when dealing with protein identification. Although rigorous use of lab coats and gloves can reduce keratin introduction into the sample, even very small quantities become a significant problem when dealing with samples of limited availability, containing very low amounts of protein, as for fossil samples. All MS/MS data obtained from fragment ions in this study (modern, medieval and fossil) were checked against keratin sequences where masses matched those of keratin tryptic peptides Modern and medieval samples The MS data from the digested medieval and clinical enamel extracts possessed masses corresponding to peptides formed from both specific and unspecific cleavage of enamel proteins (Fig. 1). Ion intensities from one recurrent peptide, observed in all the samples, which matched the mass of a peptide produced from the partial tryptic cleavage of the X isoform of TRAP (i.e. one end of this Fig. 1. MALDI mass spectra of tryptic digests of the enamel protein extracts from a) a clinical and b) a medieval sample. Only the ion with m/z 1306 from both extracts was intense enough to perform CID MS, but peaks corresponding to tryptic fragments and unspecific cleavage of enamel proteins were observed in both spectra.

3 1760 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 2. CID product ion spectra of peptide Trp-25-Pro-34 from a) clinical and b) medieval human samples as well as c) a synthetic peptide. The precursor ion [M þ H] þ is represented by the peak at the highest m/z (1306.6). Peptide sequence ions produced on cleavage of peptide bonds are labeled using Roepstorff nomenclature (Roepstorff, 2000). peptide has been formed from tryptic cleavage, the other from unspecific cleavage), were sufficiently high enough to attempt sequencing using the CID (Collision-Induced Dissociation) product ion MS to confirm its identity (Fig. 2A and B). The sequence was further confirmed by a synthetic peptide (Fig. 2C), which displayed a very similar fragmentation pattern with respect to m/z values and relative intensities above m/z 150. Most importantly, the y 4 and b 6 ions dominated all the spectra, being indicative for this sequence even at very low peptide amounts Fossil samples Both the Scladina and the Lakonis Neanderthal MALDI mass spectra contained peptides following tryptic digest of the extracts (Fig. 3). As can be seen in Fig. 3, the Scladina extract contained a great deal more protein than that of the enamel extract from the Lakonis Neanderthal. A search against collagen digest peptides was also performed for this extract using the FindPept tool, as the large number of peaks in the spectra suggests the presence of collagen contamination. As with the clinical and medieval samples, CID MS was attempted on some of the masses to confirm identity. The TRAP sequenced in the medieval and clinical extracts at m/z was not present in either of the Neanderthal extracts; however the Scladina extract did contain a peak at m/z (Fig. 3). This peptide had the greatest ion intensity of any other in the Scladina extract and was intense enough for sequencing using CID MS (Fig. 4A). The peak at m/z could correspond to a tryptic fragment of human keratin (SwissProt P35527), however the TRAP better matched the product ion data, assuming that the mass increase of 1 u was present in the N-terminal part of the sequence

4 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 3. MALDI mass spectra of tryptic digests of Neanderthal enamel protein extracts from a) Scladina and b) Lakonis. The enamel extract from Scladina, the much better preserved fossil, contains peaks corresponding to human collagen tryptic fragments. Both fossil extracts contain peaks corresponding to tryptic fragments and unspecific cleavage of human enamel proteins, but none of these were able to be conclusively identified using CID MS. A peak at m/z 842.5, matching trypsin autolysis was also observed in both spectra, indicating that protein levels in both extracts were very low. when the presumed b 6 -ion was also shifted by 1 u. Thus we hypothesized that the ion at m/z might have resulted from desamidation of the glutamine in position three of the TRAP. Following analysis using Data Explorer, both the desamidated TRAP WYESIRPYP and the keratin peptide IKFEMEQNLR were synthesized on solid phase. As peptide fragmentation in tandem mass spectrometry depends on both the ionization/fragmentation conditions, i.e. parameters selected on a specific instrument, and the peptide sequence, it is possible to confirm or disprove a given sequence by comparing the tandem mass spectra of the native peptide versus synthetic peptides. In other words, the tandem mass spectra of the synthetic peptide should display not only the same b- and y-ions but their relative intensities should also match. Thus the fragment ions detected at m/z and confirmed the TRAP sequence (Fig. 4). At the same time it clearly disproves the possibility that this is a keratin sequence, although two small signals at m/z 175 and 530 may indicate a minor contamination with the human keratin sequence. The proposed desamidation of the glutamine could be due either to the mutation of this position in the Neanderthal genome, or chemical desamidation occurring in the fossils in the burial environment. Peptides with m/z corresponding to unspecific cleavage of enamel proteins and human collagen (alpha-1(i) chain) were also detected in the Neanderthal extracts, but were too weak for successful CID MS. 3. Discussion That enamel proteins can be successfully extracted from modern mature enamel has already been confirmed by earlier studies (Porto et al., 2006), but these studies also confirm that the recoverable amount of protein from enamel in the later maturation stages is very low. The MS and MS/MS data obtained from the medieval and clinical samples in this study suggest that extraction and sequencing of proteins from mature tooth enamel using MALDI-TOF/TOF MS is possible. The quantities recovered were very limited, and only one peptide was able to be confirmed using CID MS, but this result verified the presence of a fragment of the TRAP X isoform using both the Data Explorer software (Applied Biosystems) and CID MS on three synthetic peptides. When submitted to a MASCOT MS/MS ion search of the NCBInr database, however, no significant hits were found for the ion at m/z The sequenced peptide does not appear to have been produced wholly from tryptic digestion, but may have been partially formed from enamel maturation processes, or during the extraction procedure. Though a single peptide may be searched using MASCOT MS/MS ion search, this search tool is much more powerful when analysing MS/MS runs containing data from multiple peptides, and therefore a combination of unspecific cleavage and limited MS/MS data may be hindering identification. Other masses in the spectra, not able to be sequenced, did match up with peptides formed from unspecific cleavage of enamel proteins (enamelin, ameloblastin, LRAP), and in some cases these masses corresponded to tryptic fragments from these proteins; however as no CID MS could be performed we cannot yet be certain that these proteins were present in the enamel extracts and further work is necessary to confirm their presence. The results from the Neanderthal samples are encouraging. Despite the likelihood that collagen contamination from dentine is present in the Scladina extract, the CID MS data strongly suggest that an enamel protein, namely a peptide from the TRAP X isoform, is present in sequencable amounts. The Lakonis sample is in a much worse preservation state, yet peaks, which do correspond to fragments of human enamel proteins, are present in the MALDI mass spectra. Although more research is necessary, these preliminary

5 1762 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) Fig. 4. CID product ion spectra of the precursor ion m/z from a) Scladina, b) the synthesized peptide Trp-25-Pro-34 (Gln27Glu) from the human TRAP X isoform and c) the synthesized peptide Ile-241-Arg-254 from human keratin (P35527 SwissProt) to see which produced the most complete ion series. The ion series consistent with b) Trp-Tyr-Glu- Ser-Ile-Arg-Pro-Pro-Tyr-Pro, from the synthesized TRAP produced the best fit and only two minor m/z signals at 175 and 530 might indicate very minor impurities from the keratin sequence. Peptide sequence ions produced on cleavage of peptide bonds are labeled by using Roepstorff nomenclature (Roepstorff, 2000). data suggest that proteins recovered from fossil enamel of significant age (i.e. z100,000 years) can be extracted, purified and sequenced. It is not yet clear to us why the TRAP (Trp-25-Pro-34) is the most dominant ion in the MALDI mass spectra of the protein extracts analysed. Amelogenin is the most abundant protein in enamel and during maturation proteolysis causes the formation of polypeptide fragments namely TRAP and LRAP, so it may simply be that TRAP is the most abundant peptide present in mature enamel, and therefore it dominates the MALDI mass spectra. However, we have not yet seen any evidence that TRAP is present in its whole form in the enamel extracts. No other tryptic peptides of TRAP were

6 C.M. Nielsen-Marsh et al. / Journal of Archaeological Science 36 (2009) observed in the MALDI mass spectra and the peptide sequenced is only, apparently, partially formed from tryptic digestion. That it is identifiable as the X isoform is not surprising. The X isoform is z10 more abundant than the Y isoform (Salido et al., 1992), and is therefore much more likely to be ionized and sequenced. We have never observed the Y isoform in our extracts, despite having analysed male samples. However, if the Y isoform could be recovered and sequenced, it suggests the intriguing possibility of using TRAP from fossil enamel extracts to sex fossil specimens. 4. Methods Three modern, clinical human teeth (molars, all male), one medieval human tooth from the UK (sex unknown) and two Neanderthal teeth; one from Lakonis, Greece, dating to approximately 40,000 years old (Harvati et al., 2003) and the other from Scladina Cave, Belgium dating to z100,000 years old (Toussaint and Pirson, 2006), were analysed. For the clinical and medieval samples, tooth fragments were cleaned and powdered (Dremel micro-drill) and the Manly Hodge separation method was used to separate dentine from enamel (Manly and Hodge, 1939). In this centrifugal method a mixture containing 91 volume % bromoform and 9 volume % acetone provides the optimum density (2.7 g ml 1 ) for separation of enamel and dentine. Careful separation of the enamel and dentine is crucial to the successful extraction and sequencing of enamel proteins, as any collagen remaining in the dentine could swamp the ionisation of the much less abundant enamel peptides present, hindering identification and sequencing using MALDI-TOF/TOF MS. For the Neanderthal samples, enamel was removed from the tooth by drilling (Dremel micro-drill) and care was taken to avoid the enamel dentine junction where possible, particularly in the case of the Scladina specimen, a well preserved fossil which yielded both collagen and DNA in earlier studies (Bocherens et al., 1999). We used minimum sample sizes (z2 mg of enamel); therefore the results reported for the Neanderthals are for one extraction per sample. Following drilling and enamel dentine separation, a modified version of the method described in Porto et al. (2006) was used on all samples to extract the enamel proteins. z1 2 mg of enamel powder was placed in a 0.5 ml eppendorf and 10% trichloroacetic acid (TCA) (1 mg enamel powder 200 ml 1 TCA) containing a set of protease inhibitors (Complete Mini; Roche Applied Sciences) was added. After 1 h, 2 ml of sodium deoxycholate (20 mg ml 1 )was added and the samples were stored at 4 C for 2 3 days and then centrifuged (4 C, 3,000 g, 45 min). Following centrifugation the protein pellet was washed with acetone (200 ml, 20 C) and centrifuged (4 C, 13,000 g, 5 min), this wash step was repeated and the sample was dried (CentriVap, 10 min). An in-solution digest was then performed; the pellet was re-suspended in 100 ml of 25mM ammonium bicarbonate (ABC, ph 8.0), 5 ml of 200 mm dithiothreitol (DTT) in 25 mm ABC was then added and the sample was vortexed and incubated (95 C, 10 min). 5 ml of 200 mm iodoacetamide (IAA) in 25 mm ABC was added, the sample vortexed and incubated in the dark for 45 min. A further 20 ml of 200 mm DTT in 25 mm ABC was then added and the samples were left to stand (45 min, RT). The samples were digested with trypsin (5 ml) (Promega) overnight at 37 C, after which 1% trifluoroacetic acid (TFA) was added to stop the reaction. The samples were concentrated down to 10 ml (CentriVap) and ZipTips (Millipore) were then used to further concentrate the protein extracts and remove any salts. Following extraction, digestion and purification the samples were plated onto a MALDI target and sequencing was performed via a combination of peptide mass mapping and Collision-Induced Dissociation (CID) product ion MS of selected peptides using an Applied Biosystems 4700 Proteomics Analyzer (Applied Biosystems GmbH, Darmstadt, Germany). The spectra were analysed with the Data Explorer software package (Version 4.6, Applied Biosystems GmbH). Acknowledgements We would like to thank the Max Planck Gesellschaft for funding, Corinna Sykora for advice and technical assistance with the MALDI- TOF/TOF MS, Raquel Gerlach with assistance with tooth preparation, and David Singer for the synthesis of three peptides. References Bocherens, H., Billiou, D., Mariotti, A., Palaeoenvironmental and palaeodietary implications of isotopic biogeochemistry of last interglacial Neanderthal and mammal bones in Scladina Cave (Belgium). J. Archaeol. Sci. 26, Brookes, S.J., Robinson, C., Kirkham, J., Bonass, W.A., Biochemistry and molecular biology of amelogenin proteins of developing dental enamel. Arch. Oral Biol. 40, Dean, M.C., Tooth microstructure tracks the pace of human life-history evolution. Proc. Royal Soc. B. 273, Fincham, A., Moradian-Oldak, J., Simmer, J., The structural biology of the developing dental enamel. J. Struct. Biol. 126, Glimcher, M., Brickley-Parsons, D., Levine, P., Studies of enamel proteins during maturation. Calcif. Tissue Res. 24, Glimcher, M., Cohensolal, L., Dericqles, A., Biochemical analyses of fossil enamel and dentin. Paleobiology 16, Harvati, K., Panagopoulou, E., Karkanas, P., First Neanderthal remains from Greece: the evidence from Lakonis. J. Hum. Evol. 45, Koch, P., Isotopic reconstruction of past continental environments. Annu. Rev. Earth Planet. Sci. 26, Lalueza-Fox, C., Krause, J., Caramelli, D., Catalano, G., Milani, L., Sampietro, M., Calafell, F., Martinez-Maza, C., Bastir, M., Garcia-Tabernero, A., de la Rasilla, M., Fortea, J., Paabo, S., Bertranpetit, J., Rosas, A., Mitochondrial DNA of an Iberian Neandertal suggests a population affinity with other European Neandertals. Curr. Biol. 16, R629 R630. Lee-Thorp, J., Sponheimer, M., Contributions of biogeochemistry to understanding hominin dietary ecology. Yearb. Phys. Anthropol. 4, Manly, R., Hodge, H., Density and refractive index studies of dental hard tissues: I Methods for separation and determination of purity. J. Dent. Res. 18, Nielsen-Marsh, C., Ostrom, P., Gandhi, H., Shapiro, B., Cooper, A., Collins, M., Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older that 55 ka. Geology 30, Nielsen-Marsh, C., Richards, M., Hauschka, P., Thomas-Oates, J., Trinkaus, E., Pettitt, P., Karavanic, I., Poinar, H., Collins, M., Osteocalcin protein sequences of Neanderthals and modern primates. Proc. Natl. Acad. Sci. U.S.A 102, Ostrom, P., Gandhi, H., Strahler, J., Walker, A., Andrews, P., Leykam, J., Stafford Jr., T., Kelly, R., Walker, D., Buckley, M., Humpula, J., Unravelling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse. Geochim. Cosmochim. Acta 70, Passey, B., Cerling, T., In situ stable isotope analysis (d 13 C, d 18 O) of very small teeth using laser ablation GC/IRMS. Chem. Geol. 235, Porto, I., Line, S., Laurre, H., Gerlach, R., Comparison of three methods for enamel protein extraction in different developmental phases of rat lower incisors. Eur. J. Oral Sci. 114, Roepstorff, P., MALDI-TOF mass spectrometry in protein chemistry. In: Jolles, P., Jornvall, H. (Eds.), Proteomics in Functional Genomics. Birkhauser- Verlag, Basel, pp Salido, E., Yen, P., Koprivnikar, K., Yu, L.-C., Shapiro, L., The human enamel protein gene amelogenin is expressed from both the X and Y chromosomes. Am. J. Hum. Genet. 50, Smith, T., Tafforeau, P., Reid, D., Grun, R., Eggins, S., Boutakiout, M., Hublin, J.-J., Earliest evidence of modern human life history in North African early Homo sapiens. Proc. Natl. Acad. Sci. U.S.A 104, Toussaint, M., Pirson, S., Neandertal studies in Belgium: Period Biol. 108,

Lecture 3. Tandem MS & Protein Sequencing

Lecture 3. Tandem MS & Protein Sequencing Lecture 3 Tandem MS & Protein Sequencing Nancy Allbritton, M.D., Ph.D. Department of Physiology & Biophysics 824-9137 (office) nlallbri@uci.edu Office- Rm D349 Medical Science D Bldg. Tandem MS Steps:

More information

2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry

2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry Dr. Sanjeeva Srivastava 1. Fundamental of Mass Spectrometry Role of MS and basic concepts 2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry 2 1 MS basic concepts Mass spectrometry - technique

More information

Improve Protein Analysis with the New, Mass Spectrometry- Compatible ProteasMAX Surfactant

Improve Protein Analysis with the New, Mass Spectrometry- Compatible ProteasMAX Surfactant Improve Protein Analysis with the New, Mass Spectrometry- Compatible Surfactant ABSTRACT Incomplete solubilization and digestion and poor peptide recovery are frequent limitations in protein sample preparation

More information

Sequence Identification And Spatial Distribution of Rat Brain Tryptic Peptides Using MALDI Mass Spectrometric Imaging

Sequence Identification And Spatial Distribution of Rat Brain Tryptic Peptides Using MALDI Mass Spectrometric Imaging Sequence Identification And Spatial Distribution of Rat Brain Tryptic Peptides Using MALDI Mass Spectrometric Imaging AB SCIEX MALDI TOF/TOF* Systems Patrick Pribil AB SCIEX, Canada MALDI mass spectrometric

More information

Biological Mass spectrometry in Protein Chemistry

Biological Mass spectrometry in Protein Chemistry Biological Mass spectrometry in Protein Chemistry Tuula Nyman Institute of Biotechnology tuula.nyman@helsinki.fi MASS SPECTROMETRY is an analytical technique that identifies the chemical composition of

More information

Mass Spectrometry. - Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications

Mass Spectrometry. - Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications - Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications Adapted from Mass Spectrometry in Biotechnology Gary Siuzdak,, Academic Press 1996 1 Introduction

More information

Biomolecular Mass Spectrometry

Biomolecular Mass Spectrometry Lipids ot different than other organic small molecules Carbohydrates Polymers of monosaccharides linked via glycosidic bonds (acetals/ ketals) many different combinationsvery interesting no time ucleic

More information

Trypsin Mass Spectrometry Grade

Trypsin Mass Spectrometry Grade 058PR-03 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Trypsin Mass Spectrometry Grade A Chemically Modified, TPCK treated, Affinity Purified

More information

Peptide sequencing using chemically assisted fragmentation (CAF) and Ettan MALDI-ToF Pro mass spectrometry

Peptide sequencing using chemically assisted fragmentation (CAF) and Ettan MALDI-ToF Pro mass spectrometry application note Ett MALDI-ToF Pro Peptide sequencing using chemically assisted fragmentation (CAF) d Ett MALDI-ToF Pro mass spectrometry Key words: MALDI-ToF, PSD, peptide sequencing, chemically assisted

More information

In-Solution Digestion for proteomics

In-Solution Digestion for proteomics In-Solution Digestion for proteomics Guidelines for sample preparation (How to protect your samples from contamination with keratin) 1. Try to avoid any contact of samples and solutions with dust, skin

More information

MALDI-TOF. Introduction. Schematic and Theory of MALDI

MALDI-TOF. Introduction. Schematic and Theory of MALDI MALDI-TOF Proteins and peptides have been characterized by high pressure liquid chromatography (HPLC) or SDS PAGE by generating peptide maps. These peptide maps have been used as fingerprints of protein

More information

Supporting information

Supporting information Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2014 Supporting information Glycan Reductive Isotope-coded Amino Acid Labeling (GRIAL) for Mass Spectrometry-based

More information

Biological Mass Spectrometry. April 30, 2014

Biological Mass Spectrometry. April 30, 2014 Biological Mass Spectrometry April 30, 2014 Mass Spectrometry Has become the method of choice for precise protein and nucleic acid mass determination in a very wide mass range peptide and nucleotide sequencing

More information

Fibers and extracellular matrix of hard tissues - Collagen and non-collagen proteins in hard tissues

Fibers and extracellular matrix of hard tissues - Collagen and non-collagen proteins in hard tissues Fibers and extracellular matrix of hard tissues - Collagen and non-collagen proteins in hard tissues Dr. Gábor Varga Department of Oral Biology February, 2016 Radiograph of teeth remarkable harmony of

More information

Introduction to Peptide Sequencing

Introduction to Peptide Sequencing Introduction to Peptide equencing Quadrupole Ion Traps tructural Biophysics Course December 3, 2014 12/8/14 Introduction to Peptide equencing - athan Yates 1 Why are ion traps used to sequence peptides?

More information

Comparison of mass spectrometers performances

Comparison of mass spectrometers performances Comparison of mass spectrometers performances Instrument Mass Mass Sensitivity resolution accuracy Quadrupole 1 x 10 3 0.1 Da* 0.5-1.0 pmol DE-MALDI 2 x 10 4 20 ppm 1-10 fmol peptide 1-5 pmol protein Ion

More information

Protein Identification and Phosphorylation Site Determination by de novo sequencing using PepFrag TM MALDI-Sequencing kit

Protein Identification and Phosphorylation Site Determination by de novo sequencing using PepFrag TM MALDI-Sequencing kit Application Note Tel: +82-54-223-2463 Fax : +82-54-223-2460 http://www.genomine.com venture ldg 306 Pohang techno park Pohang, kyungbuk, Korea(ROK) Protein Identification and Phosphorylation Site Determination

More information

Double charge of 33kD peak A1 A2 B1 B2 M2+ M/z. ABRF Proteomics Research Group - Qualitative Proteomics Study Identifier Number 14146

Double charge of 33kD peak A1 A2 B1 B2 M2+ M/z. ABRF Proteomics Research Group - Qualitative Proteomics Study Identifier Number 14146 Abstract The 2008 ABRF Proteomics Research Group Study offers participants the chance to participate in an anonymous study to identify qualitative differences between two protein preparations. We used

More information

(III) MALDI instrumentation

(III) MALDI instrumentation Dr. Sanjeeva Srivastava (I) Basics of MALDI-TF (II) Sample preparation In-gel digestion Zip-tip sample clean-up Matrix and sample plating (III) MALDI instrumentation 2 1 (I) Basics of MALDI-TF Analyte

More information

SUPPLEMENTAL INFORMATION

SUPPLEMENTAL INFORMATION SUPPLEMENTAL INFORMATION EXPERIMENTAL PROCEDURES Tryptic digestion protection experiments - PCSK9 with Ab-3D5 (1:1 molar ratio) in 50 mm Tris, ph 8.0, 150 mm NaCl was incubated overnight at 4 o C. The

More information

Nature Methods: doi: /nmeth.3177

Nature Methods: doi: /nmeth.3177 Supplementary Figure 1 Characterization of LysargiNase, trypsin and LysN missed cleavages. (a) Proportion of peptides identified in LysargiNase and trypsin digests of MDA-MB-231 cell lysates carrying 0,

More information

Cahn - Ingold - Prelog system. Proteins: Evolution, and Analysis Lecture 7 9/15/2009. The Fischer Convention (1) G (2) (3)

Cahn - Ingold - Prelog system. Proteins: Evolution, and Analysis Lecture 7 9/15/2009. The Fischer Convention (1) G (2) (3) Chapter 4 (1) G Proteins: Evolution, and Analysis Lecture 7 9/15/2009 A V L I M P F W Chapter 4 (2) S (3) T N Q Y C K R H D E The Fischer Convention Absolute configuration about an asymmetric carbon related

More information

Mass Spectrometry. Mass spectrometer MALDI-TOF ESI/MS/MS. Basic components. Ionization source Mass analyzer Detector

Mass Spectrometry. Mass spectrometer MALDI-TOF ESI/MS/MS. Basic components. Ionization source Mass analyzer Detector Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio (limit

More information

TECHNICAL BULLETIN. R 2 GlcNAcβ1 4GlcNAcβ1 Asn

TECHNICAL BULLETIN. R 2 GlcNAcβ1 4GlcNAcβ1 Asn GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit Product Code PP0201 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Glycosylation is one of the most common posttranslational

More information

SUPPORTING INFORMATION. Lysine Carbonylation is a Previously Unrecognized Contributor. to Peroxidase Activation of Cytochrome c by Chloramine-T

SUPPORTING INFORMATION. Lysine Carbonylation is a Previously Unrecognized Contributor. to Peroxidase Activation of Cytochrome c by Chloramine-T Electronic Supplementary Material (ESI) for Chemical Science. This journal is The Royal Society of Chemistry 2019 SUPPORTING INFORMATION Lysine Carbonylation is a Previously Unrecognized Contributor to

More information

Nature Methods: doi: /nmeth Supplementary Figure 1

Nature Methods: doi: /nmeth Supplementary Figure 1 Supplementary Figure 1 Subtiligase-catalyzed ligations with ubiquitin thioesters and 10-mer biotinylated peptides. (a) General scheme for ligations between ubiquitin thioesters and 10-mer, biotinylated

More information

Problem-solving Test: The Mechanism of Protein Synthesis

Problem-solving Test: The Mechanism of Protein Synthesis Q 2009 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 37, No. 1, pp. 58 62, 2009 Problem-based Learning Problem-solving Test: The Mechanism

More information

Rapid Detection and Identification of Bacteria in Milk Products by Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry (MALDI-MS/MS)

Rapid Detection and Identification of Bacteria in Milk Products by Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry (MALDI-MS/MS) Rapid Detection and Identification of Bacteria in Milk Products by Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry (MALDI-MS/MS) Nyesa Enakaya It is essential to be able to identify

More information

Automated Sample Preparation/Concentration of Biological Samples Prior to Analysis via MALDI-TOF Mass Spectroscopy Application Note 222

Automated Sample Preparation/Concentration of Biological Samples Prior to Analysis via MALDI-TOF Mass Spectroscopy Application Note 222 Automated Sample Preparation/Concentration of Biological Samples Prior to Analysis via MALDI-TOF Mass Spectroscopy Application Note 222 Joan Stevens, Ph.D.; Luke Roenneburg; Tim Hegeman; Kevin Fawcett

More information

Protein sequence mapping is commonly used to

Protein sequence mapping is commonly used to Reproducible Microwave-Assisted Acid Hydrolysis of Proteins Using a Household Microwave Oven and Its Combination with LC-ESI MS/MS for Mapping Protein Sequences and Modifications Nan Wang and Liang Li

More information

The distribution of log 2 ratio (H/L) for quantified peptides. cleavage sites in each bin of log 2 ratio of quantified. peptides

The distribution of log 2 ratio (H/L) for quantified peptides. cleavage sites in each bin of log 2 ratio of quantified. peptides Journal: Nature Methods Article Title: Corresponding Author: Protein digestion priority is independent of their abundances Mingliang Ye and Hanfa Zou Supplementary Figure 1 Supplementary Figure 2 The distribution

More information

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow Purpose Described herein is a workflow that combines the isobaric tagging reagents, itraq Reagents, with the separation power

More information

Phosphorylation of proteins Steve Barnes Feb 19th, 2002 in some cases, proteins are found in a stable, hyperphosphorylated state, e.g.

Phosphorylation of proteins Steve Barnes Feb 19th, 2002 in some cases, proteins are found in a stable, hyperphosphorylated state, e.g. Phosphorylation of proteins Steve Barnes Feb 19th, 2002 in some cases, proteins are found in a stable, hyperphosphorylated state, e.g., casein more interestingly, in most other cases, it is a transient

More information

LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program

LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program

More information

Supporting Information. Lysine Propionylation to Boost Proteome Sequence. Coverage and Enable a Silent SILAC Strategy for

Supporting Information. Lysine Propionylation to Boost Proteome Sequence. Coverage and Enable a Silent SILAC Strategy for Supporting Information Lysine Propionylation to Boost Proteome Sequence Coverage and Enable a Silent SILAC Strategy for Relative Protein Quantification Christoph U. Schräder 1, Shaun Moore 1,2, Aaron A.

More information

Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Supporting Information

Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Supporting Information Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry M. Montana Quick, Christopher M. Crittenden, Jake A. Rosenberg, and Jennifer S. Brodbelt

More information

PHOSPHOPEPTIDE ANALYSIS USING IMAC SAMPLE PREPARATION FOLLOWED BY MALDI-MS and MALDI PSD MX

PHOSPHOPEPTIDE ANALYSIS USING IMAC SAMPLE PREPARATION FOLLOWED BY MALDI-MS and MALDI PSD MX PHOSPHOPEPTIDE ANALYSIS USING IMAC SAMPLE PREPARATION FOLLOWED BY MALDI-MS and MALDI PSD MX E. Claude 1, E. Emekwue 2, M. Snel 1, T. McKenna 1 and J. Langridge 1 1: Waters Corporation, Manchester, UK 2:

More information

This exam consists of two parts. Part I is multiple choice. Each of these 25 questions is worth 2 points.

This exam consists of two parts. Part I is multiple choice. Each of these 25 questions is worth 2 points. MBB 407/511 Molecular Biology and Biochemistry First Examination - October 1, 2002 Name Social Security Number This exam consists of two parts. Part I is multiple choice. Each of these 25 questions is

More information

AccuMAP Low ph Protein Digestion Kits

AccuMAP Low ph Protein Digestion Kits TECHNICAL MANUAL AccuMAP Low ph Protein Digestion Kits Instruc ons for Use of Products VA1040 and VA1050 5/17 TM504 AccuMAP Low ph Protein Digestion Kits All technical literature is available at: www.promega.com/protocols/

More information

Trypsin Digestion Mix

Trypsin Digestion Mix G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name 239PR Trypsin Digestion Mix Provides optimal buffered conditions for in gel trypsin digestion

More information

Mass Spectrometry and Proteomics - Lecture 4 - Matthias Trost Newcastle University

Mass Spectrometry and Proteomics - Lecture 4 - Matthias Trost Newcastle University Mass Spectrometry and Proteomics - Lecture 4 - Matthias Trost Newcastle University matthias.trost@ncl.ac.uk previously Peptide fragmentation Hybrid instruments 117 The Building Blocks of Life DNA RNA Proteins

More information

PosterREPRINT A NOVEL APPROACH TO MALDI-TOF-MS SAMPLE PREPARATION. Presented at ABRF 2002, Austin, Texas, USA, 9th - 12th March 2002.

PosterREPRINT A NOVEL APPROACH TO MALDI-TOF-MS SAMPLE PREPARATION. Presented at ABRF 2002, Austin, Texas, USA, 9th - 12th March 2002. Introduction A NOVEL APPROACH TO MALDI-TOF-MS SAMPLE PREPARATION Ed Bouvier 2, Jeff Brown 1, Emmanuelle Claude 1, John L. Gebler 2, Weibin Chen 2, *Dominic Gostick 1, Kevin Howes 1, James Langridge 1,

More information

Chemical Biology, Option II Mechanism Based Proteomic Tagging Case History CH1

Chemical Biology, Option II Mechanism Based Proteomic Tagging Case History CH1 Proteome Wide Screening of Serine Protease Activity Proc Natl Acad Sci 1999, 97, 14694; Proteomics 2001, 1, 1067; Proc Natl Acad Sci 2002, 99, 10335; Biochemistry 2001, 40, 4005; J. Am. Chem. Soc., 2005,

More information

ProteaseMAX Surfactant, Trypsin Enhancer

ProteaseMAX Surfactant, Trypsin Enhancer Technical Bulletin ProteaseMAX Surfactant, Trypsin Enhancer INSTRUCTIONS FOR USE OF PRODUCTS V2071 AND V2072. PRINTED IN USA. Revised 1/10 ProteaseMAX Surfactant, Trypsin Enhancer All technical literature

More information

Supporting Information

Supporting Information Supporting Information Cyclic Peptidyl Inhibitors against Human Peptidyl-Prolyl Isomerase Pin1 Tao Liu, Yu Liu, Hung-Ying Kao, *,, and Dehua Pei Table of Contents: Table S1. Structures of amino acid building

More information

Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials

Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials Supporting information 1 2 3 Volume 71 (2015) Supporting information for article: 4 5 6 7 8 Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for

More information

Enhancing Sequence Coverage in Proteomics Studies by Using a Combination of Proteolytic Enzymes

Enhancing Sequence Coverage in Proteomics Studies by Using a Combination of Proteolytic Enzymes Enhancing Sequence Coverage in Proteomics Studies by Using a Combination of Proteolytic Enzymes Dominic Baeumlisberger 2, Christopher Kurz 3, Tabiwang N. Arrey, Marion Rohmer 2, Carola Schiller 3, Thomas

More information

REDOX PROTEOMICS. Roman Zubarev.

REDOX PROTEOMICS. Roman Zubarev. REDOX PROTEOMICS Roman Zubarev Roman.Zubarev@ki.se Physiological Chemistry I, Department for Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm What is (RedOx) Proteomics? Proteomics -

More information

PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System

PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System Application Note LC/MS PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System Purpose This application note describes an automated workflow

More information

Quantitative LC-MS/MS Analysis of Glucagon. Veniamin Lapko, Ph.D June 21, 2011

Quantitative LC-MS/MS Analysis of Glucagon. Veniamin Lapko, Ph.D June 21, 2011 Quantitative LC-MS/MS Analysis of Glucagon Veniamin Lapko, Ph.D June 21, 2011 Contents Comparison with small molecule LC-MS/MS LC-MS/MS sensitivity of peptides detection Stability: neat vs. matrix solutions

More information

Chapter 10. Regulatory Strategy

Chapter 10. Regulatory Strategy Chapter 10 Regulatory Strategy Regulation of enzymatic activity: 1. Allosteric Control. Allosteric proteins have a regulatory site(s) and multiple functional sites Activity of proteins is regulated by

More information

Three Dimensional Mapping and Imaging of Neuropeptides and Lipids in Crustacean Brain

Three Dimensional Mapping and Imaging of Neuropeptides and Lipids in Crustacean Brain Three Dimensional Mapping and Imaging of Neuropeptides and Lipids in Crustacean Brain Using the 4800 MALDI TOF/TOF Analyzer Ruibing Chen and Lingjun Li School of Pharmacy and Department of Chemistry, University

More information

Microalbuminuric Diabetic patients N=18

Microalbuminuric Diabetic patients N=18 ONLINE APPENDIX Table A1 Clinical and laboratory features of healthy subjects, type 2 diabetic patients and NDCKD patients enrolled in the present study. Healthy Subjects N= 2 Normoalbuminuric Diabetic

More information

The Detection of Allergens in Food Products with LC-MS

The Detection of Allergens in Food Products with LC-MS The Detection of Allergens in Food Products with LC-MS Something for the future? Jacqueline van der Wielen Scope of Organisation Dutch Food and Consumer Product Safety Authority: Law enforcement Control

More information

Intro to Physical Anthropology. Content: Chapter 1

Intro to Physical Anthropology. Content: Chapter 1 Intro to Physical Anthropology Content: Chapter 1 1 Course website https://creason.co/ Very important for this class -Syllabus -Assignment instructions -Sample essays, tests, and questions -Study guides

More information

LAB#23: Biochemical Evidence of Evolution Name: Period Date :

LAB#23: Biochemical Evidence of Evolution Name: Period Date : LAB#23: Biochemical Evidence of Name: Period Date : Laboratory Experience #23 Bridge Worth 80 Lab Minutes If two organisms have similar portions of DNA (genes), these organisms will probably make similar

More information

Electronic Supplementary Information

Electronic Supplementary Information Electronic Supplementary Information Microwave-assisted Kochetkov amination followed by permanent charge derivatization: A facile strategy for glycomics Xin Liu a,b, Guisen Zhang* a, Kenneth Chan b and

More information

Ion Source. Mass Analyzer. Detector. intensity. mass/charge

Ion Source. Mass Analyzer. Detector. intensity. mass/charge Proteomics Informatics Overview of spectrometry (Week 2) Ion Source Analyzer Detector Peptide Fragmentation Ion Source Analyzer 1 Fragmentation Analyzer 2 Detector b y Liquid Chromatography (LC)-MS/MS

More information

Glycosylation analysis of blood plasma proteins

Glycosylation analysis of blood plasma proteins Glycosylation analysis of blood plasma proteins Thesis booklet Eszter Tóth Doctoral School of Pharmaceutical Sciences Semmelweis University Supervisor: Károly Vékey DSc Official reviewers: Borbála Dalmadiné

More information

Properties of amino acids in proteins

Properties of amino acids in proteins Properties of amino acids in proteins one of the primary roles of DNA (but far from the only one!!!) is to code for proteins A typical bacterium builds thousands types of proteins, all from ~20 amino acids

More information

Agilent Protein In-Gel Tryptic Digestion Kit

Agilent Protein In-Gel Tryptic Digestion Kit Agilent 5188-2749 Protein In-Gel Tryptic Digestion Kit Agilent Protein In-Gel Tryptic Digestion Kit Instructions Kit Contents The Protein In-Gel Tryptic Digestion Kit includes sufficient reagents for approximately

More information

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples:

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Dr. Sanjeeva Srivastava IIT Bombay Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Sample preparation for serum proteome analysis Sample

More information

Enamel thickness in Asian human canines and premolars

Enamel thickness in Asian human canines and premolars ANTHROPOLOGICAL SCIENCE Vol. 118(3), 191 198, 2010 Brief Communication Enamel thickness in Asian human canines and premolars Robin N.M. FEENEY 1, John P. ZERMENO 2, Donald J. REID 3, Syozi NAKASHIMA 4,

More information

The 1997 ABRF Mass Spectrometry Committee Collaborative Study: Identification of Phosphopeptides in a Tryptic Digest of Apomyoglobin

The 1997 ABRF Mass Spectrometry Committee Collaborative Study: Identification of Phosphopeptides in a Tryptic Digest of Apomyoglobin The 1997 ABRF Mass Spectrometry Committee Collaborative Study: Identification of Phosphopeptides in a Tryptic Digest of Apomyoglobin Kristine Swiderek, Farzin Gharahdaghi, Lowell Ericsson, Murray Hackett,

More information

Small Molecule Drug Imaging of Mouse Tissue by MALDI-TOF/TOF Mass Spectrometry and FTMS

Small Molecule Drug Imaging of Mouse Tissue by MALDI-TOF/TOF Mass Spectrometry and FTMS Bruker Daltonics Application Note # MT-93/FTMS-38 Small Molecule Drug Imaging of Mouse Tissue by MALDI-TOF/TOF Mass Spectrometry and FTMS Introduction Matrix Assisted Laser Desorption Ionization (MALDI)

More information

Proteomics of body liquids as a source for potential methods for medical diagnostics Prof. Dr. Evgeny Nikolaev

Proteomics of body liquids as a source for potential methods for medical diagnostics Prof. Dr. Evgeny Nikolaev Proteomics of body liquids as a source for potential methods for medical diagnostics Prof. Dr. Evgeny Nikolaev Institute for Biochemical Physics, Rus. Acad. Sci., Moscow, Russia. Institute for Energy Problems

More information

Quantitation of Protein Phosphorylation Using Multiple Reaction Monitoring

Quantitation of Protein Phosphorylation Using Multiple Reaction Monitoring Quantitation of Protein Phosphorylation Using Multiple Reaction Monitoring Application Note Authors Ning Tang, Christine A. Miller and Keith Waddell Agilent Technologies, Inc. Santa Clara, CA USA This

More information

MS/MS Scan Modes. Eötvös University, Budapest April 16, MS/MS Scan Modes. Árpád Somogyi. Product Ion Scan Select. Scan. Precursor Ion Scan Scan

MS/MS Scan Modes. Eötvös University, Budapest April 16, MS/MS Scan Modes. Árpád Somogyi. Product Ion Scan Select. Scan. Precursor Ion Scan Scan MS/MS Modes Árpád Somogyi Eötvös University, Budapest April 16, 2012 MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) 1 modes in a triple quadrupole (QqQ) (one quadrupole

More information

Advances in Hybrid Mass Spectrometry

Advances in Hybrid Mass Spectrometry The world leader in serving science Advances in Hybrid Mass Spectrometry ESAC 2008 Claire Dauly Field Marketing Specialist, Proteomics New hybrids instruments LTQ Orbitrap XL with ETD MALDI LTQ Orbitrap

More information

[ Care and Use Manual ]

[ Care and Use Manual ] MALDI Calibration Kit I. Introduction The MALDI Calibration Kit is a conveniently packaged selection of MALDI matrices and calibration standards (includes high-purity Neg Ion Mode Calibrant for accurate

More information

Chemical Mechanism of Enzymes

Chemical Mechanism of Enzymes Chemical Mechanism of Enzymes Enzyme Engineering 5.2 Definition of the mechanism 1. The sequence from substrate(s) to product(s) : Reaction steps 2. The rates at which the complex are interconverted 3.

More information

Universal sample preparation method for proteome analysis

Universal sample preparation method for proteome analysis nature methods Universal sample preparation method for proteome analysis Jacek R Wi niewski, Alexandre Zougman, Nagarjuna Nagaraj & Matthias Mann Supplementary figures and text: Supplementary Figure 1

More information

BIOCHEMISTRY REVIEW. Overview of Biomolecules. Chapter 4 Protein Sequence

BIOCHEMISTRY REVIEW. Overview of Biomolecules. Chapter 4 Protein Sequence BIOCHEMISTRY REVIEW Overview of Biomolecules Chapter 4 Protein Sequence 2 3 4 Are You Getting It?? A molecule of hemoglobin is compared with a molecule of lysozyme. Which characteristics do they share?

More information

SYNAPT G2-S High Definition MS (HDMS) System

SYNAPT G2-S High Definition MS (HDMS) System SYNAPT G2-S High Definition MS (HDMS) System High performance, versatility, and workflow efficiency of your MS system all play a crucial role in your ability to successfully reach your scientific and business

More information

Brief Communication: Dental Development and Enamel Thickness in the Lakonis Neanderthal Molar

Brief Communication: Dental Development and Enamel Thickness in the Lakonis Neanderthal Molar AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 138:112 118 (2009) Brief Communication: Dental Development and Enamel Thickness in the Lakonis Neanderthal Molar T.M. Smith, 1 * K. Harvati, 1 A.J. Olejniczak,

More information

Shotgun Proteomics MS/MS. Protein Mixture. proteolysis. Peptide Mixture. Time. Abundance. Abundance. m/z. Abundance. m/z 2. Abundance.

Shotgun Proteomics MS/MS. Protein Mixture. proteolysis. Peptide Mixture. Time. Abundance. Abundance. m/z. Abundance. m/z 2. Abundance. Abundance Abundance Abundance Abundance Abundance Shotgun Proteomics Protein Mixture 1 2 3 MS/MS proteolysis m/z 2 3 Time µlc m/z MS 1 m/z Peptide Mixture m/z Block Diagram of a Mass Spectrometer Sample

More information

Quantitative chromatin proteomics reveals a dynamic histone. post-translational modification landscape that defines asexual

Quantitative chromatin proteomics reveals a dynamic histone. post-translational modification landscape that defines asexual Quantitative chromatin proteomics reveals a dynamic histone post-translational modification landscape that defines asexual and sexual Plasmodium falciparum parasites Nanika Coetzee 1, Simone Sidoli 2,

More information

Sequence Coverage (%) Profilin-1 P UD 2

Sequence Coverage (%) Profilin-1 P UD 2 Protein Name Accession Number (Swissprot) Sequence Coverage (%) No. of MS/MS Queries Mascot Score 1 Reference Cytoskeletal proteins Beta-actin P60709 37 14 298 Alpha-actin P68032 33 10 141 20 Beta-actin-like

More information

Collagenase Assay Kit

Collagenase Assay Kit Collagenase Assay Kit Catalog # 31 and 32 For Research Use Only - Not Human or Therapeutic Use INTRODUCTION The collagenases are members of the matrix metalloproteinase (MMP) family and degrade collagen

More information

Taiwan. University, Hualien 970, Taiwan

Taiwan. University, Hualien 970, Taiwan Glutathione-Bound Gold Nanoclusters for Selective-Binding and Detection of Glutathione S-Transferase-Fusion Proteins from Cell Lysates Cheng-Tai Chen, 1 Wei-Jen Chen, 1 Chao-Zong Liu, 2,3 Ling-Ya Chang,

More information

PosterREPRINT AN AUTOMATED METHOD TO SELF-CALIBRATE AND REJECT NOISE FROM MALDI PEPTIDE MASS FINGERPRINT SPECTRA

PosterREPRINT AN AUTOMATED METHOD TO SELF-CALIBRATE AND REJECT NOISE FROM MALDI PEPTIDE MASS FINGERPRINT SPECTRA Overview AN AUTOMATED METHOD TO SELF-CALIBRATE AND REJECT NOISE FROM MALDI PEPTIDE MASS FINGERPRINT SPECTRA Jeffery M Brown, Neil Swainston, Dominic O. Gostick, Keith Richardson, Richard Denny, Steven

More information

MALDI Imaging Drug Imaging Detlev Suckau Head of R&D MALDI Bruker Daltonik GmbH. December 19,

MALDI Imaging Drug Imaging Detlev Suckau Head of R&D MALDI Bruker Daltonik GmbH. December 19, MALDI Imaging Drug Imaging Detlev Suckau Head of R&D MALDI Bruker Daltonik GmbH December 19, 2014 1 The principle of MALDI imaging Spatially resolved mass spectra are recorded Each mass signal represents

More information

Mass Spectrometry Infrastructure

Mass Spectrometry Infrastructure Mass Spectrometry Infrastructure Todd Williams, Ph.D. Director KU Mass Spectrometry and Analytical Proteomics Laboratory Mass Spectrometry Lab B025 Malott Hall Mission The Mass Spectrometry and analytical

More information

Chapter 3. Protein Structure and Function

Chapter 3. Protein Structure and Function Chapter 3 Protein Structure and Function Broad functional classes So Proteins have structure and function... Fine! -Why do we care to know more???? Understanding functional architechture gives us POWER

More information

1. Sample Introduction to MS Systems:

1. Sample Introduction to MS Systems: MS Overview: 9.10.08 1. Sample Introduction to MS Systems:...2 1.1. Chromatography Interfaces:...3 1.2. Electron impact: Used mainly in Protein MS hard ionization source...4 1.3. Electrospray Ioniztion:

More information

Supporting Information for: Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler, Annegret Binas,

Supporting Information for: Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler, Annegret Binas, Supporting Information for: Oncocin (VDKPPYLPRPRPPRRIYNR-NH 2 ): a novel antibacterial peptide optimized against Gram-negative human pathogens Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler,

More information

Collagenase Types 1 through 7

Collagenase Types 1 through 7 COLLAGENASE Tissue Dissociation/Cell Isolation PRODUCT HIGHLIGHTS Clostridium histolyticum contains two distinct but related genes for collagenase. The col G gene codes for a 936 amino acid protein designated

More information

Proteins: Proteomics & Protein-Protein Interactions Part I

Proteins: Proteomics & Protein-Protein Interactions Part I Proteins: Proteomics & Protein-Protein Interactions Part I Jesse Rinehart, PhD Department of Cellular & Molecular Physiology Systems Biology Institute DNA RNA PROTEIN DNA RNA PROTEIN Proteins: Proteomics

More information

Walking upright Specific changes in chewing design: teeth, jaws and skull. Homonoidea, Hominidae, Hominininae, Hominini, Hominina, Homo

Walking upright Specific changes in chewing design: teeth, jaws and skull. Homonoidea, Hominidae, Hominininae, Hominini, Hominina, Homo Bio 1M: Hominins (complete) 1 Emergence Hominins refer to people and our upright ancestors Characterized by: Walking upright Specific changes in chewing design: teeth, jaws and skull Taxonomy Homonoidea,

More information

Electronic Supplementary Information. Table of Contents

Electronic Supplementary Information. Table of Contents Electronic Supplementary Information Examination of native chemical ligation using peptidyl prolyl thioester Takahiro Nakamura, Akira Shigenaga, Kohei Sato, Yusuke Tsuda, Ken Sakamoto, and Akira Otaka*

More information

Types of Modifications

Types of Modifications Modifications 1 Types of Modifications Post-translational Phosphorylation, acetylation Artefacts Oxidation, acetylation Derivatisation Alkylation of cysteine, ICAT, SILAC Sequence variants Errors, SNP

More information

Chapter 13 Dental Development and Age at Death of a Middle Paleolithic Juvenile Hominin from Obi-Rakhmat Grotto, Uzbekistan

Chapter 13 Dental Development and Age at Death of a Middle Paleolithic Juvenile Hominin from Obi-Rakhmat Grotto, Uzbekistan Chapter 13 Dental Development and Age at Death of a Middle Paleolithic Juvenile Hominin from Obi-Rakhmat Grotto, Uzbekistan Tanya M. Smith, Donald J. Reid, Anthony J. Olejniczak, Shara Bailey, Mica Glantz,

More information

Proteomics Grade. Protocol. Catalog # Agilent Technologies. Research Use Only. Not for use in Diagnostic Procedures. Version A, January 2010

Proteomics Grade. Protocol. Catalog # Agilent Technologies. Research Use Only. Not for use in Diagnostic Procedures. Version A, January 2010 Proteomics Grade Trypsin Catalog #204310 Protocol Version A, January 2010 Research Use Only. Not for use in Diagnostic Procedures. Agilent Technologies Notices Agilent Technologies, Inc. 2010 No part of

More information

A Study of Peptide Peptide Interaction by Matrix-Assisted Laser Desorption/Ionization

A Study of Peptide Peptide Interaction by Matrix-Assisted Laser Desorption/Ionization A Study of Peptide Peptide Interaction by Matrix-Assisted Laser Desorption/Ionization Amina S. Woods and Marilyn A. Huestis Chemistry and Drug Metabolism, NIDA Intramural Research Program, NIDA, NIH, Baltimore,

More information

Mass Spectrometry and Proteomics. Professor Xudong Yao Bioanalytical Chemistry Spring 2007

Mass Spectrometry and Proteomics. Professor Xudong Yao Bioanalytical Chemistry Spring 2007 Mass Spectrometry and Proteomics Professor Xudong Yao Bioanalytical Chemistry Spring 2007 Proteomics and -omics Roles of mass spectrometry Comparative proteomics Chemical proteomics Protein, Proteome and

More information

SUPPLEMENTARY MATERIAL

SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus Colocynthis Muhammad Bashir Khan, 1,3 Hidayatullah khan, 2 Muhammad

More information

Analysis of Peptides via Capillary HPLC and Fraction Collection Directly onto a MALDI Plate for Off-line Analysis by MALDI-TOF

Analysis of Peptides via Capillary HPLC and Fraction Collection Directly onto a MALDI Plate for Off-line Analysis by MALDI-TOF Analysis of Peptides via Capillary HPLC and Fraction Collection Directly onto a MALDI Plate for Off-line Analysis by MALDI-TOF Application Note 219 Joan Stevens, PhD; Luke Roenneburg; Kevin Fawcett (Gilson,

More information

In-Gel Tryptic Digestion Kit

In-Gel Tryptic Digestion Kit INSTRUCTIONS In-Gel Tryptic Digestion Kit 3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105 89871 1468.2 Number Description 89871 In-Gel Tryptic Digestion Kit, sufficient reagents for approximately

More information

N-terminal charge-driven de novo sequencing by using ASDF-incorporated Curved Field Reflectron

N-terminal charge-driven de novo sequencing by using ASDF-incorporated Curved Field Reflectron PO-CON1534E N-terminal charge-driven de novo sequencing by using ASDF-incorporated Curved Field Reflectron ASMS 2015 ThP 361 Yuzo Yamazaki 1 ; Keisuke Shima 1 ; Atsushi Kitanaka 2 ; Masahiro Miyashita

More information

MALDI Imaging Mass Spectrometry

MALDI Imaging Mass Spectrometry MALDI Imaging Mass Spectrometry Nan Kleinholz Mass Spectrometry and Proteomics Facility The Ohio State University Mass Spectrometry and Proteomics Workshop What is MALDI Imaging? MALDI: Matrix Assisted

More information