Mass spectrometry in protein identification/characterization

Size: px
Start display at page:

Download "Mass spectrometry in protein identification/characterization"

Transcription

1 Mass spectrometry in protein identification/characterization A comprehensive investigation on any protein system requires an integrated study of several aspects, including: (1) identification (primary to quaternary structures) (2) changes in protein levels across different states (3) determination of post-translational modifications (4) identification of functionally interactive partners These aspects are currently the object of a complex network of different disciplines (genetics, biochemistry, chemistry, informatics) known as proteomics. Proteome: the complete set of proteins coded by a genome (IUPAC Compendium of Chemical Terminology, 2005)

2 Proteomics: the main goals Protein Localization Quantification Identification Proteomics Structure Modifications Function Activity

3 The initial stage in the study of a protein is the knowledge of its structure at different levels: As for the primary structure, a typical proteomic method is the so-called bottom-up approach: the protein, eventually separated from other proteins, is first digested with a protease (like trypsin), then the mixture of its peptides is analyzed by MS, and eventually MS/MS, in order to recognize their molecular weights (Peptide Mass Fingerprint, PMF) and their aminoacid sequence from fragmentation spectra. Primary structure is finally reconstructed using bioinformatics.

4 Proteomics: separations preliminary to MS analysis spot cut Enzymatic digestion (Trypsin) Peptide mixture 2D polyacrylamide-gel electrophoresis (2D-PAGE) Protein elution from gel MS, MS/MS, MS n analysis Peptide mixture Mono-dimensional liquid chromatography

5 Several enzymes, each one with specific sites of hydrolysis, are currently available for protein digestion: example

6 A more complex separating strategy for digestive peptides mixtures is on line/off line bi-dimensional liquid chromatography: In the first dimension (IEX = Ion EXchange) peptide separation is based on charge: A) step-wise cation (Na + /K + ) gradient elution (on line mode) Poly-sulphoethyl-aspartamide B) continuous gradient elution (off line mode)

7 Influence of peptide charge on IEX separation retention times: peptide mixture obtained from digestion of a whole proteome (yeast extract): S. Gygi, Harvard Medical School

8 On-line bidimensional liquid chromatography: IEX (SCX)-RP Besides an ion exchange (Strong Cation exchanger, SCX) and a RP chromatography column, on line coupling between IEX and RP HPLC requires: two separate pumping systems and mobile phases, a 6 port valve, a 10 port valve, a C18 enrichment column. A typical 2D separation for tryptic digests consists of two subsequent stages: 1) The sample is loaded in the SCX column through the 6-port valve. The SCX eluate is then transferred into a C18 enrichment column, mounted on the 10-port valve. Tryptic peptides are captured by the enrichment column, while the salt used for SCX separation is discarded to waste (desalting).

9 2) The 10-port valve is switched, so that the RP mobile phase, pumped by the 2 nd pump, enters the enrichment column and elutes the peptides towards the C18 analytical column, followed by a MS detector. Although the C18 analytical column is usually a capillary or nano-lc column, so that small fractions eluted from the SCX column can be subsequently separated, the peptide elution from the SCX column cannot be continuous. Salt steps are then used, at regular time intervals, into the SCX mobile phase: at each step the salt concentration is increased and a fraction of more retained peptides is released from the SCX column and transferred to the enrichment column. A new salt step is performed only after the enrichment-separation-analysis stage for the fraction corresponding to the previous salt step has been completed.

10 The described method, known as MudPIT (Multi dimensional Protein identification Technology), leads to as many LC-MS (MS/MS) chromatograms as the number of salt steps (i.e. fractions collected from the SCX column). LC-MS traces obtained for the tryptic digest of the whole proteome of yeast (the NaCl steps are indicated):

11 Off-line bidimensional liquid chromatography: IEX (SCX)-RP In off-line 2D-LC fractions are collected regularly from the SCX column. Each fraction is then loaded in the enrichment column for desalting and subsequently transferred in the RP column. In this case the SCX separation is performed using gradient elution: the total chromatographic resolution is higher, but also the analysis time is increased (more fractions than before are analysed by LC-MS).

12 Off line 2D LC (SCX-RP) MS analysis performed on the tryptic digest of Saccaromyces Cerevisiae proteome: UV detection can be used for a pre-evaluation of the peptide amount in each fraction collected after SCX separation.

13 A remarkable number of peptides (and then proteins) can be identified when MS and MS/MS are used for detection after the second chromatographic dimension (RPC). S. Gygi, J. Peng, Harvard Medical School

14 Network of separation-ms detection approaches in proteomics Sample Single protein Protein mixture Enzymatic digestion Enzymatic digestion Single peptide Peptide mixture LC o 2D-LC MALDI- ToF-MS MW ESI-MS MW (LC) MALDI- ToF-MS MW set LC-ESI-MS MW set MALDI- ToF-MS ESI-MS MW MALDI- ToF-ToF MS ESI-MS/MS Fragm. pattern MALDI- ToF-MS ESI-MS PMF MALDI- ToF-ToF MS ESI-MS/MS Fragm. patterns set Bioinformatics

15 MALDI-ToF-MS: relevant information for proteomics Integral protein mixture Tryptic peptide mixture Mass (m/z) Mass (m/z) Molecular weight determination (up to Da) Competition for ionization between proteins Difficult quantification Peptide Mass Fingerprinting (PMF) Competition for ionization between peptides Difficult quantification Difficult coupling with separation techniques

16 Off-line coupling between HPLC and MALDI-ToF-MS HPLC MALDI-Prep MALDI-ToF-MS The HPLC eluent is mixed with MALDI matrix and then sprayed periodically on a well of the MALDI sample plate.

17 LC-ESI-MS: separation and analysis of integral proteins Relative Abundance Pasteurized milk MS-TIC La Lg-B Lg-A Relative abundance LgB MW = Time / min m/z Relative abundance La MW = m/z Relative abundance LgA MW = m/z I. Losito, T. Carbonara, L. Monaci, F. Palmisano Anal. Bioanal. Chem. 389, 2007, 2065

18 LC-ESI-MS: separation and analysis of peptide mixtures CapLC separation of a tryptic digest or other peptide mixtures ESI-MS analysis by Q-ToF MS Small sample volumes High Resolving Power (R = ) and sensitivity (fmoles) ESI-Q-ToF-MS spectrometer with Z- spray source Autosampler CapLC Capilary C18 column(15 cm, 0.32 mm ID); flow: 5 L/min, injection volume: 4-10 L

19 An example: study of Maillard reaction on whey proteins -LgB + lactose, 70 C, 5 h CapLC-ESI-Q-ToF-MS TIC trace for the tryptic digest [M+6H] 6+ [M+2H] 2+ Estrazione M = Da [M+H] + (m/z) = corrente Th ionica su m/z Even long tryptic peptides can be detected, due to recognition of high charge states by high resolution MS.

20 Bioinformatic approaches based on MS data Enzym. digest. Peptide mixture Protein hydrolysis during natural processes (LC-)MS analysis protein (mixture) Single MW or MW set Peptide Mass Fingerprint Softwares Protein Prospector Mascot Sequest Phenyx Aldente NCBInr SwissProt UniProt Genpept Genomic/proteomic databases (db) Adapted from: Cottrell J.S., Database Searching for Protein Identification and Characterization, 5th Annual Workshop and User Meeting of the American Society of Mass Spectrometry, 2005.

21 Comparing experimental and predicted (virtual) PMF ESI Sofwares operating on PMF can perform in silico digestion, i.e. generate the presumed peptides obtained when an enzyme is used to hydrolyse a protein, for all or a part of the proteins listed in a database. A comparison is then made between experimental and virtual PMFs.

22 As shown for the Aldente PMF tool, the user has to provide, as input: the peak list (MWs of experimental peptides) the database the taxon, i.e. the organisms or group of organisms to which the searched protein is supposed to belong, if known further data on the protein, if known The tolerance on the matching between MWs of experimental and virtual peptides in the PMF has to be also specified.

23 Several candidate proteins are usually listed as output:

24 Choice among candidate proteins Peak list +TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS... a= e-004, t0= e+001, Thresholded PMF Max counts m/z, amu Best candidate database search

25 Peptides recognized are also listed for each candidate sequence: The protein sequence coverage provided by matching peptides is a key parameter to decide whether the identification is correct.

26 LC-ESI-MS/MS, MS n : identification of the aminoacid sequence of a peptide enz. digest. peptide mixture Protein hydrolysis during natural processes analysis (2D-LC or LC-MS/MS) protein (mixture) m/z = Relative abundance Softwares m/z Protein Prospector Mascot Sequest Phenyx NCBInr SwissProt UniProt Genpept Genomic/proteomic databases (db) Adapted from: Cottrell J.S., Database Searching for Protein Identification and Characterization, 5th Annual Workshop and User Meeting of the American Society of Mass Spectrometry, 2005.

27 LC-ESI-MS/MS, MS n analysis of peptides: fundamental stages Full scan MS acquisition ( Th) Processing of MS spectra for each peak Ion current extraction on specific m/z ratios MS/MS (MS 3 ) acquisitions Relative Abundance Full scan MS chromatogram (base peak) Retention time / min Relative Abundance Relative Abundance Relative Abundance m/z = m/z = m/z = Retention time / min t r = min m/z = Relative Abundance m/z Relative abundance m/z I. Losito, T. Carbonara, M.D. De Bari, M. Gobbetti, F. Palmisano, C.G. Rizzello, P.G. Zambonin, Rapid. Comm. Mass Spectrom., 20 (2006) 1-9

28 LC-ESI-MS/MS analysis: data dependent approach Most softwares currently adopted for LC-ESI-MS, MS/MS analysis of complex peptide mixtures are able to perform a Data Dependent/Directed Analysis (DDA). During chromatographic elution, a fast, explorative MS acquisition is performed first, then only ions whose intensity overcomes a userdefined threshold (up to a maximum number) are sequentially isolated and fragmented.

29 Even the ion charge state can be automatically evaluated and used as a criterion for the selection of ions to be fragmented. Charge state check Explorative MS scan MS/MS spectrum The main drawback of the DDA approach is that peak reconstruction through a specific MS event is usually not complete.

30 Peptide bond: the key of peptide fragmentation pk 1 ~ 2.2 pk 2 ~ 9.4 pk R

31 The 20 proteinogenic aminoacids: main properties

32 MS-relevant information on the 20 proteinogenic aminoacids

33 Peptide fragmentation in MS/MS Peptide bonds are the weak points of a peptide molecular structure, thus they are typically involved in peptide fragmentation during MS/MS experiments. The peptide bond breakage can leave the positive charge on a fragment containing the carboxylic end:

34 Alternatively, the positive charge can be located on a fragment containing the aminic end: Resonance stabilization

35 Occasionally, further product ions, named as x and c, containing the carboxylic or the aminic end, respectively, can be found in MS/MS spectra of peptides:

36 The generally accepted nomenclature for product ions generated during MS/MS experiments on peptides is the one proposed by Roepstorff and Fohlman in 1984 and can be summarized in the following scheme: The number used as subscript indicates how many aminoacid residues are contained in the corresponding product ion.

37 The m/z ratios of the main product ions can be easily calculated if the aminoacidic sequence is known, as shown for b and y ions of the DAEFR peptide: y 5 y 4 y 3 y 2 y 1 b 5 b 4 b 3 b 2 b 1

38 In some cases, low m/z ratio product ions can be observed in MS/MS spectra of peptides. They may arise from the aminoacidic residue at the carboxylic end, after CO 2 loss and breakage of the peptide bond with the last-but-one aminoacidic residue: Pro (Arg, Asn) 70 Val 72 Leu 86 Met 104 His 110 Phe 120 Tyr 136 Trp 159 Typical y 1 ions can be observed when tryptic peptides are considered, due to the presence of Lysine or Arginine at the carboxylic end of the peptide. Lys y Arg y b 1 ions are almost never observed in CID/CAD MS/MS spectra

39 Occasionally, internal cleavage ions can be observed during peptide MS/MS fragmentation. As an example, a b-type ion including the second and third aminoacidic residues, starting from the aminic terminus, can be observed: These ions are observed most frequently when Proline is enclosed in the peptide sequence. In this case P is the first amino acid (from the left end) of the internal fragment. An internal fragment with just a single side chain formed by a combination of a-type and y-type cleavage is called an immonium ion:

40 Immonium ions are labelled with the 1 letter code for the corresponding amino acid: As shown in the table, further fragments can be formed from immonium ions (those having higher intensities are shown with bold characters).

41 An example of MS/MS spectrum interpretation: [Glu] 1 fibrinopeptide B MW [M+2H] 2+ The y-ions series seems to predominate in this case but the y 13 ion, implying a breakage of the first peptide bond from the left end (amino terminus) is not observed

42 Special approaches to peptide fragmentation The identification of peptide sequences from MS/MS data has recently benefited from new approaches developed for precursor ions fragmentation. Pulsed Q Dissociation These approach, available on the LTQ Linear Ion Trap, is based on the application of a very short (100 s) resonance excitation pulse when the precursor ion has a high q value (according to the Mathieu diagram). The Main RF Voltage is lowered (thus lowering the q values for precursor and product ions) only after a delay time, i.e. when fragmentation actually begins.

43 As shown for the QNCDQFEK tryptic peptide from BSA (doubly charged ion), Pulsed Q Dissociation enables generation and detection also of peptide product ions that either are difficult to form with CID or have low m/z ratios (b 1 and y 1 and immonium ions): In this example the presence of ions with m/z 145 and is due to preliminary peptide derivatization with the itraq TM reagents, developed by Applied Biosystems for relative/absolute protein quantification.

44 itraq (isobaric Tags for Relative/Absolute Quantitation) reagents are four isobaric compounds whose structure is made by a Reporter, a Balance and a Peptide Reactive Group: Each of them is linked covalently, through its PRG, to the NH 2 groups of a peptide (i.e. the N-terminus and those of lysines, if present). During MS/MS fragmentation, a common fragment, at m/z 145, is observed for all the reagents and a specific fragment, i.e. the Reporter Group, is observed for each of them.

45 In the itraq methodology up to four samples, different (i.e. arising from different treatments or physiological/pathological states) but containing the same protein(s) of interest, are subjected, in a parallel scheme, to enzymatic digestion, then digestive peptides are labelled with a different itraq reagent, according to the sample. The labelled digests are then combined and analyzed by LC- MS/MS (after a cleaning-up or separation stage). Under the assumption that the yields of digestion and labelling processes are not significantly different in the four cases, the relative intensity of itraq reporter ions in the MS/MS spectrum of a specific peptide will provide information on the abundance of the corresponding protein in the two samples.

46 High energy Collisional Dissociation (HCD) The LTQ-Orbitrap spectrometer enables alternative approaches to fragmentation of precursor ions selected by the LIT, exploiting either the C- trap or a special octapolar collision cell, located behind the C-Trap (the cell is optional for some models):

47 The C-trap is commonly used to transfer ions orthogonally towards the Orbitrap, yet it can be used also as a collision cell if ions are accelerated into the trap and the latter is preliminarily filled with gas (Pressure < 1 mtorr): high energy trapping leads to HCD MS/MS spectra. As with Pulsed Q Dissociation, peptide fragment ions with low m/z ratios are enhanced in C-traprelated MS/MS spectra. Moreover, y-type ions are favored. V V Low energy trapping High energy trapping P <1 mtorr

48

49 HCD can be performed also in a special octapolar collision cell (Oct 2 in the following scheme) located behind the C-trap and housed in a gastight container, with an internal pressure of about mbar: The Oct 2 DC offset decides the collisional energy involved in HCD fragmentation (HCD CE). After generation, fragment ions can be transferred back to the C-trap and then to the Orbitrap.

50 In this example the enhancement of low m/z ratio signals is observed in the HCD MS/MS spectrum of a peptide contained in a mouse cell digest, labelled with the itraq reagents: A peculiar feature of this MS/MS spectrum is the only partial overlap of b- type and y-type ion series.

51 Post translational modifications (PTMs) Definition: any modification occurring on a protein after its translation from the genetic code. Such modifications can regulate function, activity state, cellular location and dynamic interactions with other proteins of the translated protein. Main facts about PTMs: more than 200 modification types currently known occurring on at least 80% of eucatiotic proteins located at specific sequence motifs (sequons) often transient usually present at sub-stoichiometric levels often acting together

52 Phosphorylation is a typical PTM, occurring on aminoacidic residues having an OH group. A HO-P(O) 2 - group transfer from a ATP molecule to a protein is catalyzed by a kinase; the dephosphorylation of a protein is catalyzed by a phosphatase:

53 MS and MS/MS analysis, along with appropriate extraction/separation techniques, play a key role in identifying phosphorylation sites:

54 Graphic reconstruction of a 2D-PAGE gel relevant to brain proteins (real dimensions: cm)

55 LC-MS chromatographic traces relevant to Lys-C digests of spots #246 and 247. The most relevant differences between the two traces are marked by arrows: The 80 u difference between marked peaks could correspond to a phosphate group.

56 The MS/MS spectrum obtained on the digestive peptide enables an identification of the corresponding sequence, showing the phosphorylation site:

57 MALDI imaging MALDI-ToF-MS (MS/MS) can be exploited for tissue section imaging, i.e. to evaluate the abundance and distribution of molecules of biomedical significance (e.g. proteins, peptides, lipids, metabolites, drugs) In this case the MALDI matrix is applied on the frozen tissue section by casting or spraying. Special mapping softwares can provide MS images of the sample, starting from MALDI spectra obtained using raster scanning.

58 An example of imaging MALDI-ToF-MS: location of different proteins in a rat brain section A 20 m lateral resolution has been recently achieved in MALDI imaging using a special laser beam for ionization.

59 Surface Enhanced Laser Desorption Ionization (SELDI) In the SELDI method, protein-matrix solutions are applied to the spots of ProteinChip Arrays, special multi-well sample stages, derivatized with planar chromatographic chemistries or modified with biomolecules capable of molecular recognition (e.g. antibodies). The proteins actively interact with the array surface and become sequestered according to their surface interaction potential as well as separated from salts and other sample contaminants by subsequent on-spot washing with appropriate buffer solutions:

60 After analyte extraction the Protein Chip array is mounted in a MALDIlike spectrometer, whose laser is then fired sequentially over each spot, using a raster map as reference. Mass spectra obtained from single laser shots are averaged to provide a SELDI MS spectrum for each spot: The choice of arrays with spots having different specificity towards sample proteins can reduce the complexity of MALDI spectra and the risks due to competition for ionization. The more refined control on the interactions between the sample and the sample stage surface usually leads to an improvement in quantitation reliability.

61 In this example, SELDI-MS is used to evaluate the kinetics of dephosphorylation of peptide ISpYGRKKRRQRRRP using alkaline phosphatase:

62 Lipidomics Lipidomics is an emerging discipline strictly related to Proteomics and to other omic disciplines. Its main goals are the total analysis of lipids in a cell, organ or organism and, at a more refined level, the study of their organization, functions and interactions with other biomolecules. Genomics Lipidomics Proteomics Glycomics Metabolomics Adapted from X. Wang, University of Missouri

63 Studies made in the last two decades have shown that lipids play a much more complex role than simple energy storage in living systems. Cell membranes Pathologies Lipidomics Energy storage Cell regulation Lipidomics studies have then relevance for several aspects, including pathologies like diabetes, degenerative diseases and even cancer.

64 Lipids: a classification on a molecular basis Adapted from: D.W. Ball et alt., The Basics of General, Organic and Biological Chemistry, v 1.0, Chapter 17

65 Lipids architecture in the cell membrane According to the fluid mosaic model, the currently accepted model of cell membranes, supramolecular interactions of special lipids (phospho/sphyngo/glyco-lipids and cholesterol) between themselves and other biomolecules (proteins, oligosaccharides) are determinant for the cell membrane functioning.

66 extracellular matrix Cholesterol cytoplasma Glycero-phospholipids Glycolipids

67 The distribution of phospholipids (PLs) is not homogenous neither in the two membrane leaflets nor in the membranes of different cells (the w/w % averaged on all the membrane is shown). (SM) (PC) (PE) (PS) Remarkable differences are observed between eucariotyc and procariotyc cells: Membrane Cholest. PC SM PE PI PS PG CL Glycolip. Myelin Erythrocyte ,5 15 2, Hepatocyte Mitochondr. (Internal) , E. coli PG = phosphatidyl-glycerol, CL = cardiolypines

68 The (poly)unsaturated fatty acids linked to glycerol in phospholipids play a fundamental role in terms of membrane fluidity: H C O N H P leaflet C=C They usually have cis C=C bonds, that: modify the side chain packing in the two leaflets lower the temperature of crystalline phase transition increase the membrane fluidity

69 Palmitic (16:0) Stearic (18:0) Oleic (18:1) Linoleic (18:2) Linolenic (18:3) Arachidonic (20:4) DHA* (22:6) Others Incidence of unsaturated fatty acids in PL Human erythrocites Egg Bovine brain fatty acids DHA = DocosaHexaenoic Acid % in total fatty acids

70 Structural alterations of phospholipid side chains The in vivo reaction between Reactive Oxygen Species (ROS) and C=C bonds along side chains of phospholypids leads to oxidized species that can alter dramatically the lipid packing and, consequently, the cell membrane functionality.

71 Lipid extraction: the Bligh and Dyer protocol for PLs CH3OH: 2.5 vol Vortexing Centrifugation 5000 rpm, 5 min Separation of the protein pellet CHCl3: 1.25 vol Sample suspension in buffer: 1 vol Homogeneous suspension 1. Collection of the bottom chloroform phase, containing phospholipids 2. Solvent evaporation under N 2 3. Re-dissolution into pure CHCl 3 (known volume) Subsequent additions (with homogeneization) 4. Phosphorous quantification 5. Solvent evaporation on aliquots and storage under N 2 at -28 C CHCl3: 1.25 vol H 2 O/NaCl: 1.25 vol Transfer of the surnatant to a separatory funnel E. G. Bligh, W. J. Dyer Can. J. Biochem. Phys., 37 (1959)

72 Possible approaches to phospholipid extract analysis Sub-class separation by NPC or TLC on silica Sample shotgun lipidomics RP-HPLC (HILIC)-ESI- MS, MS/MS analysis MALDI-ToF- MS analysis RP-HPLC (HILIC)-ESI- MS, MS/MS analysis MW set Fragm. patterns set MALDI- ToF-MS direct analysis MW set ESI-MS, MS/MS direct analysis MW set Fragm. patterns set MW set MW set Fragm. patterns set Bioinformatics

73 Shotgun lipidomics: MALDI-ToF-MS Positive ion MALDI-ToF spectrum of phospholipids extracted from human Low Density Lipoproteins (LDL). Matrix: 2,5-dihydroxy-benzoic acid J. Schiller et al., Anal. Biochem. 309 (2002)

74 Shotgun lipidomics: ESI-MS Acyl-carnitines GalC: GalactoCerebrosides FFA: Free Fatty Acids X. Han, R.W. Gross J. Lip. Res. 44 (2003)

75 Shotgun lipidomics: ESI-MS X. Han, R.W. Gross J. Lip. Res. 44 (2003)

76 Separation of phospholipid classes: Thin Layer Chromatography (TLC) Stationary phase: silica gel Mobile phase: chloroform, ethyl-acetate, acetone, isopropanol, ethanol, methanol, water, acetic acid (30:6:6:6:16:28:6:2) Run time: 55 min Samples: A: mixture of standard phospholipids B1,B2: phospholipids of blood microparticels from healthy donors Start L-PX = Lyso-phospholipids A.M. Weerheim et al., Anal. Biochem. 302 (2002)

77 Separation of phospholipid classes: Normal Phase Chromatography (NPC) Stationary phase: silica Mobile phase: A- hexane, 2-propanol, potassium acetate (ph 7.0), ethanol, glacial acetic acid (367:490:62:100:0.6) B - hexane, 2-propanol, potassium acetate (ph 7.0), acetonitrile, glacial acetic acid (442:490:62:25:0.6) E.J. Lesnefsky et al. Anal. Biochem., 285 (2000)

78 Separation of phospholipids based on side chain hydrophobicity: Reverse Phase Chromatography (RPC) extracted Ion Chromatograms (XIC) Stationary phase: C18 Mobile phase (isocratic elution): acetonitrile/methanol/ triethyl-amine (550/1000/25 v/v) Detection: (+) ESI-MS Sample: mixture of phosphatidylcholines extracted from hen egg The retention on the C18 stationary phase is controlled primarily by the acyl chain on the sn-1 position of glycerol E.A.A.M. Vernooij et al. J. Sep. Sci., 25 (2002)

79 RPC separation of complex lipid mixtures Relative Abundance PC(16:0/18:2) PC(16:0/18:1) Time (min) RPC-ESI-3D Ion Trap-MS Total Ion Current (TIC) chromatograms obtained for the PL extracts of erythrocitary membrane from two healthy donors. I. Losito et al., data collected in the SMART Centre

80 Separation of phospholipid classes: Hydrophilic Interaction Liquid Chromatography (HILIC) HILIC combines the features of the main liquid chromatography techniques (Normal Phase, NP; Ion Chromatography, IC; Reverse Phase, RP): silica diol cyano amino B. Buszewski and S. Noga, Anal. Bional. Chem., 402 (2012)

81 Relatively polar organic solvents, miscible with water, are used in the HILIC mobile phase. Acetonitrile and methanol are the most common (like in RPC). Such a choice gives HILIC full compatibility with ESI-MS. Water percentage in the HILIC mobile phase is usually lower than 20% (v/v). Under these conditions the retention is thought to be based on analyte partitioning between water-reach layers adsorbed on the polar stationary phase and acetonitrile (methanol)-enriched mobile phase. B. Buszewski and S. Noga, Anal. Bional. Chem., 402 (2012)

82 R 1 R2 HILIC separation of complex lipid mixtures HILIC-ESI-IT-ToF-MS analyses of lipid extracts from parts of Arabidopsis Thaliana MGDG DGDG HILIC is able to separate lipid classes, usually providing narrower peaks than NPC or RPC for each species. SQDG Y. Okazaki, Metabolomics, 9 (2013) S121-S131

83 HILIC-ESI-MS analysis of PL extracts: evidence for class-related separation Blood MP sample PS PE PC SM Lyso-PC Platelet sample (same donor) Blood MP Sample RPC separation I. Losito et al., Anal. Chem. 85 (2013)

84 extracted Ion Current (XIC) chromatograms TIC ESI(+) Relative Abundance PC(16:0/0:0) PC(18:0/0:0) PC(18:1/0:0) SM(d18:1/16:0) SM(d18:1/24:1) SM(d16:1/16:0) PC(16:0/18:3) PC(16:0/20:4) PC(18:0/18:1) PC(18:1/20:4) PC(18:0/20:4) Time (min) I. Losito et al., Anal. Chem. 85 (2013)

4th Multidimensional Chromatography Workshop Toronto (January, 2013) Herman C. Lam, Ph.D. Calibration & Validation Group

4th Multidimensional Chromatography Workshop Toronto (January, 2013) Herman C. Lam, Ph.D. Calibration & Validation Group 4th Multidimensional Chromatography Workshop Toronto (January, 2013) Herman C. Lam, Ph.D. Calibration & Validation Group MDLC for Shotgun Proteomics Introduction General concepts Advantages Challenges

More information

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

LC/MS Method for Comprehensive Analysis of Plasma Lipids

LC/MS Method for Comprehensive Analysis of Plasma Lipids Application Note omics LC/MS Method for Comprehensive Analysis of Plasma s Authors Tomas Cajka and Oliver Fiehn West Coast Metabolomics Center, University of California Davis, 451 Health Sciences Drive,

More information

Mass Spectrometry based metabolomics

Mass Spectrometry based metabolomics Mass Spectrometry based metabolomics Metabolomics- A realm of small molecules (

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

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

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

The use of mass spectrometry in lipidomics. Outlines

The use of mass spectrometry in lipidomics. Outlines The use of mass spectrometry in lipidomics Jeevan Prasain jprasain@uab.edu 6-2612 utlines Brief introduction to lipidomics Analytical methodology: MS/MS structure elucidation of phospholipids Phospholipid

More information

Introduction to Proteomics 1.0

Introduction to Proteomics 1.0 Introduction to Proteomics 1.0 CMSP Workshop Pratik Jagtap Managing Director, CMSP Objectives Why are we here? For participants: Learn basics of MS-based proteomics Learn what s necessary for success using

More information

LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES. Gilles Frache Materials Characterization Day October 14 th 2016

LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES. Gilles Frache Materials Characterization Day October 14 th 2016 LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES Gilles Frache Materials Characterization Day October 14 th 2016 1 MOLECULAR ANALYSES Which focus? LOCALIZATION of molecules by Mass Spectrometry

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

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

Relative Quantitation of Human Polymorphonuclear Leukocyte Cell Membrane GPEtn Lipids

Relative Quantitation of Human Polymorphonuclear Leukocyte Cell Membrane GPEtn Lipids Relative Quantitation of Human Polymorphonuclear Leukocyte Cell Membrane GPEtn Lipids Using the QTRAP System with mtraq Reagents Karin A. Zemski-Berry 1, John M. Hevko 2, and Robert C. Murphy 1 1 Department

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

Impact of Chromatography on Lipid Profiling of Liver Tissue Extracts

Impact of Chromatography on Lipid Profiling of Liver Tissue Extracts Impact of Chromatography on Lipid Profiling of Liver Tissue Extracts Application Note Clinical Research Authors Mark Sartain and Theodore Sana Agilent Technologies, Inc. Santa Clara, California, USA Introduction

More information

Phospholipid characterization by a TQ-MS data based identification scheme

Phospholipid characterization by a TQ-MS data based identification scheme P-CN1716E Phospholipid characterization by a TQ-MS data based identification scheme ASMS 2017 MP-406 Tsuyoshi Nakanishi 1, Masaki Yamada 1, Ningombam Sanjib Meitei 2, 3 1 Shimadzu Corporation, Kyoto, Japan,

More information

LC-Based Lipidomics Analysis on QTRAP Instruments

LC-Based Lipidomics Analysis on QTRAP Instruments LC-Based Lipidomics Analysis on QTRAP Instruments Junhua Wang and Paul RS Baker SCIEX LC-Based Lipidomics Analysis Topics Covered Lipid extraction techniques Hydrophilic Interaction Chromatography (HILIC)

More information

Metabolomics: quantifying the phenotype

Metabolomics: quantifying the phenotype Metabolomics: quantifying the phenotype Metabolomics Promises Quantitative Phenotyping What can happen GENOME What appears to be happening Bioinformatics TRANSCRIPTOME What makes it happen PROTEOME Systems

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

SUPPLEMENTARY DATA. Materials and Methods

SUPPLEMENTARY DATA. Materials and Methods SUPPLEMENTARY DATA Materials and Methods HPLC-UV of phospholipid classes and HETE isomer determination. Fractionation of platelet lipid classes was undertaken on a Spherisorb S5W 150 x 4.6 mm column (Waters

More information

MASS SPECTROMETRY BASED METABOLOMICS. Pavel Aronov. ABRF2010 Metabolomics Research Group March 21, 2010

MASS SPECTROMETRY BASED METABOLOMICS. Pavel Aronov. ABRF2010 Metabolomics Research Group March 21, 2010 MASS SPECTROMETRY BASED METABOLOMICS Pavel Aronov ABRF2010 Metabolomics Research Group March 21, 2010 Types of Experiments in Metabolomics targeted non targeted Number of analyzed metabolites is limited

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 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

Lipids Analysis. Lipids

Lipids Analysis. Lipids Lipids Analysis Stephen Barnes 3 5 15 Lipids Lipids are mostly very hydrophobic Most are conjugates of fatty acids of a variety of chain lengths, which have different degrees of unsaturation, cis trans

More information

Application of a new capillary HPLC- ICP-MS interface to the identification of selenium-containing proteins in selenized yeast

Application of a new capillary HPLC- ICP-MS interface to the identification of selenium-containing proteins in selenized yeast Application of a new capillary HPLC- ICP-MS interface to the identification of selenium-containing proteins in selenized yeast Application note Food supplements Authors Juliusz Bianga and Joanna Szpunar

More information

Using Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes

Using Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes Using Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes Eric S. Grumbach,, Diane M. Diehl and Jeffrey R. Mazzeo 2003 Waters Corp. Outline Introduction HILIC Definition

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

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

New Instruments and Services

New Instruments and Services New Instruments and Services Liwen Zhang Mass Spectrometry and Proteomics Facility The Ohio State University Summer Workshop 2016 Thermo Orbitrap Fusion http://planetorbitrap.com/orbitrap fusion Thermo

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 Supporting information Figure legends Supplementary Table 1. Specific product ions obtained from fragmentation of lithium adducts in the positive ion mode comparing the different positional isomers of

More information

2D-LC as an Automated Desalting Tool for MSD Analysis

2D-LC as an Automated Desalting Tool for MSD Analysis 2D-LC as an Automated Desalting Tool for MSD Analysis Direct Mass Selective Detection of a Pharmaceutical Peptide from an MS-Incompatible USP Method Application Note Biologics and Biosimilars Author Sonja

More information

New Instruments and Services

New Instruments and Services New Instruments and Services http://planetorbitrap.com/orbitrap fusion Combining the best of quadrupole, Orbitrap, and ion trap mass analysis in a revolutionary Tribrid architecture, the Orbitrap Fusion

More information

A Definitive Lipidomics Workflow for Human Plasma Utilizing Off-line Enrichment and Class Specific Separation of Phospholipids

A Definitive Lipidomics Workflow for Human Plasma Utilizing Off-line Enrichment and Class Specific Separation of Phospholipids A Definitive Lipidomics Workflow for Human Plasma Utilizing Off-line Enrichment and Class Specific Separation of Phospholipids Jeremy Netto, 1 Stephen Wong, 1 Federico Torta, 2 Pradeep Narayanaswamy, 2

More information

Edgar Naegele. Abstract

Edgar Naegele. Abstract Simultaneous determination of metabolic stability and identification of buspirone metabolites using multiple column fast LC/TOF mass spectrometry Application ote Edgar aegele Abstract A recent trend in

More information

Principles of Shotgun Lipidomics

Principles of Shotgun Lipidomics Principles of Shotgun Lipidomics Xianlin Han Diabetes and Obesity Research Center Sanford-Burnham Medical Research Institute Lake Nona Orlando, FL 32827 What is shotgun lipidomics? Original definition

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

Mass spectrometry Technologies in Lipid chemistry

Mass spectrometry Technologies in Lipid chemistry Mass spectrometry Technologies in Lipid chemistry Rabah Soliymani University Of Helsinki Protein Chemistry Unit Biomedicum Helsinki Rabah.soliymani@helsinki.fi Complex_&_dynamic_mixtures (few copies to

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

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

Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System

Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System Marta Kozak Clinical Research Applications Group Thermo Fisher Scientific San Jose CA Clinical Research use only, Not for

More information

Glycerolipid Analysis. LC/MS/MS Analytical Services

Glycerolipid Analysis. LC/MS/MS Analytical Services Glycerolipid Analysis LC/MS/MS Analytical Services Molecular Characterization and Quantitation of Glycerophospholipids in Commercial Lecithins by High Performance Liquid Chromatography with Mass Spectrometric

More information

Protein and peptide separations,

Protein and peptide separations, A Quarterly Technical Newsletter Spring Grace Vydac: Leading the Way in Separations for Proteomics Protein and peptide separations, always important to protein chemists and enzymologists, have recently

More information

Mass-Spectrometric Analysis of Lipids (Lipidomics)

Mass-Spectrometric Analysis of Lipids (Lipidomics) Mass-Spectrometric Analysis of Lipids (Lipidomics) 1. Identification 2. Quantification 3. Metabolism Why to do lipidomics? Biology: Functions of different lipids? Medicine: Diagnostics and Therapy Industry:

More information

Metabolomic fingerprinting of serum samples by direct infusion mass spectrometry

Metabolomic fingerprinting of serum samples by direct infusion mass spectrometry Metabolomic fingerprinting of serum samples by direct infusion mass spectrometry Raúl González-Domínguez * Department of Chemistry, Faculty of Experimental Sciences. University of Huelva, Spain. * Corresponding

More information

Learning Objectives. Overview of topics to be discussed 10/25/2013 HIGH RESOLUTION MASS SPECTROMETRY (HRMS) IN DISCOVERY PROTEOMICS

Learning Objectives. Overview of topics to be discussed 10/25/2013 HIGH RESOLUTION MASS SPECTROMETRY (HRMS) IN DISCOVERY PROTEOMICS HIGH RESOLUTION MASS SPECTROMETRY (HRMS) IN DISCOVERY PROTEOMICS A clinical proteomics perspective Michael L. Merchant, PhD School of Medicine, University of Louisville Louisville, KY Learning Objectives

More information

Analytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products)

Analytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products) Analytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products) The target compound to be determined is 2, 4, 5-T. 1. Instrument Liquid Chromatograph-tandem mass spectrometer (LC-MS/MS)

More information

The detergent-solubilized and gel filtration purified rhodopsin was partitioned against

The detergent-solubilized and gel filtration purified rhodopsin was partitioned against Supplement Jastrzebska et al. Materials and Methods The detergent-solubilized and gel filtration purified rhodopsin was partitioned against H 2 O/MeOH/CHCl 3, and the bottom layer was removed, dried down,

More information

About bioassay of Oximes:? New isolation alternatives from biomatrices? Chromatographic separation issues? Reserve on using MS or MS/MS detection

About bioassay of Oximes:? New isolation alternatives from biomatrices? Chromatographic separation issues? Reserve on using MS or MS/MS detection State of the art: xime-type AChE Reactivators: Wide use as antidotes in organo phosphorous compounds poisoning; Synthesis of new congeners; Intense studies on their transfer through BBB; Intense studies

More information

An Introduction to the Use of itraq Reagents for Amino Acid Analysis

An Introduction to the Use of itraq Reagents for Amino Acid Analysis An Introduction to the Use of itraq Reagents for Amino Acid Analysis Lisa Sapp Clinical Research and Forensic Toxicology Product Manager Mass Spectrometry Systems Amino Acid Analysis Using itraq Reagents

More information

Lipid Analysis. Andréina Laffargue, IRD CRYMCEPT Montpellier workshop, October 17th Introduction to lipid structures

Lipid Analysis. Andréina Laffargue, IRD CRYMCEPT Montpellier workshop, October 17th Introduction to lipid structures Lipid Analysis Andréina Laffargue, IRD CRYMCEPT Montpellier workshop, October 17th 2005 Introduction to lipid structures Fatty acids Acylglycerols Glycerophospholipids Sterols Strategies involved in lipid

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

NIH Public Access Author Manuscript J Proteome Res. Author manuscript; available in PMC 2014 July 05.

NIH Public Access Author Manuscript J Proteome Res. Author manuscript; available in PMC 2014 July 05. NIH Public Access Author Manuscript Published in final edited form as: J Proteome Res. 2013 July 5; 12(7): 3071 3086. doi:10.1021/pr3011588. Evaluation and Optimization of Mass Spectrometric Settings during

More information

Rapid Lipid Profiling of Serum by Reverse Phase UPLC-Tandem Quadrupole MS

Rapid Lipid Profiling of Serum by Reverse Phase UPLC-Tandem Quadrupole MS Rapid Lipid Profiling of Serum by Reverse Phase UPLC-Tandem Quadrupole MS Mark Ritchie and Evelyn Goh Waters Pacific Pte Ltd., Singapore A P P L I C AT ION B E N E F I T S Delivers a rapid 10-min MRM method

More information

High-Resolution Analysis of Intact Triglycerides by Reversed Phase HPLC Using the Agilent 1290 Infinity LC UHPLC System

High-Resolution Analysis of Intact Triglycerides by Reversed Phase HPLC Using the Agilent 1290 Infinity LC UHPLC System High-Resolution Analysis of Intact Triglycerides by Reversed Phase HPLC Using the Agilent 1290 Infinity LC UHPLC System Application Note Food, Hydrocarbon Processing Authors Michael Woodman Agilent Technologies,

More information

New Solvent Grade Targeted for Trace Analysis by UHPLC-MS

New Solvent Grade Targeted for Trace Analysis by UHPLC-MS New Solvent Grade Targeted for Trace Analysis by UHPLC-MS Subhra Bhattacharya, Deva H. Puranam, and Stephen C. Roemer Thermo Fisher Scientific Fisher Chemical, One Reagent Lane, Fair Lawn, NJ Material

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

Achieve Broad Lipid Quantitation using a High-Throughput Targeted Lipidomics Method

Achieve Broad Lipid Quantitation using a High-Throughput Targeted Lipidomics Method Achieve Broad Lipid Quantitation using a High-Throughput Targeted Lipidomics Method LC-Based Approach for Lipid Class Separation and Quantitation on QTRAP 6500+ System Mackenzie Pearson, Santosh Kumar

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

Measuring Lipid Composition LC-MS/MS

Measuring Lipid Composition LC-MS/MS Project: Measuring Lipid Composition LC-MS/MS Verification of expected lipid composition in nanomedical controlled release systems by liquid chromatography tandem mass spectrometry AUTHORED BY: DATE: Sven

More information

Comprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids

Comprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids PO-CON1576E Comprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids HPLC 2015 PSB-MULTI-06 Yoshiyuki WATABE, Tetsuo IIDA, Daisuke NAKAYAMA, Kanya TSUJII, Saki

More information

Rapid, Simple Impurity Characterization with the Xevo TQ Mass Spectrometer

Rapid, Simple Impurity Characterization with the Xevo TQ Mass Spectrometer Robert Plumb, Michael D. Jones, and Marian Twohig Waters Corporation, Milford, MA, USA INTRODUCTION The detection and characterization of impurities and degradation products of an active pharmaceutical

More information

More structural information with MS n

More structural information with MS n PRODUCT SPECIFICATIONS The LTQ XL linear ion trap mass spectrometer More structural information with MS n The LTQ XL linear ion trap mass spectrometer delivers more structural information faster and with

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

Automated Sample Preparation for Profiling Fatty Acids in Blood and Plasma using the Agilent 7693 ALS

Automated Sample Preparation for Profiling Fatty Acids in Blood and Plasma using the Agilent 7693 ALS Automated Sample Preparation for Profiling Fatty Acids in Blood and Plasma using the Agilent 7693 ALS Application Note Clinical Research Authors Frank David and Bart Tienpont, Research Institute for Chromatography,

More information

O O H. Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION EXPERIMENTAL. LC /MS conditions

O O H. Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION EXPERIMENTAL. LC /MS conditions Simplifying Qual/Quan Analysis in Discovery DMPK using UPLC and Xevo TQ MS Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION The determination of the drug metabolism

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

Choosing the metabolomics platform

Choosing the metabolomics platform Choosing the metabolomics platform Stephen Barnes, PhD Department of Pharmacology & Toxicology University of Alabama at Birmingham sbarnes@uab.edu Challenges Unlike DNA, RNA and proteins, the metabolome

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

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

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

The Raptor HILIC-Si Column

The Raptor HILIC-Si Column The Raptor HILIC-Si Column With Raptor LC columns, Restek chemists became the first to combine the speed of superficially porous particles (also known as SPP or core-shell particles) with the resolution

More information

Accurate Quantification of Lipid Species by Electrospray Ionization Mass Spectrometry Meets a Key Challenge in Lipidomics

Accurate Quantification of Lipid Species by Electrospray Ionization Mass Spectrometry Meets a Key Challenge in Lipidomics Metabolites 2011, 1, 21-40; doi:10.3390/metabo1010021 Review OPEN ACCESS metabolites ISSN 2218-1989 www.mdpi.com/journal/metabolites/ Accurate Quantification of Lipid Species by Electrospray Ionization

More information

Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP 2016

Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP 2016 Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP 2016 2015 Waters Corporation 1 Overview 1. 2D RP RP LC of peptides 2. RP-LC

More information

Increased Identification Coverage and Throughput for Complex Lipidomes

Increased Identification Coverage and Throughput for Complex Lipidomes Increased Identification Coverage and Throughput for Complex Lipidomes Reiko Kiyonami, David Peake, Yingying Huang, Thermo Fisher Scientific, San Jose, CA USA Application Note 607 Key Words Q Exactive

More information

Core E Analysis of Neutral Lipids from Human Plasma June 4, 2010 Thomas J. Leiker and Robert M. Barkley

Core E Analysis of Neutral Lipids from Human Plasma June 4, 2010 Thomas J. Leiker and Robert M. Barkley Core E Analysis of Neutral Lipids from Human Plasma June 4, 2010 Thomas J. Leiker and Robert M. Barkley This protocol describes the extraction and direct measurement of cholesterol esters (CEs) and triacylglycerols

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

Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis

Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Ying Qing Yu Waters Corporation, Milford, MA, USA APPLICATION BENEFITS

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

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

New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics

New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics New Mass Spectrometry Tools to Transform Metabolomics and Lipidomics July.3.13 Ken Miller Vice President of Marketing, Life Sciences Mass Spectrometry 1 The world leader in serving science Omics & the

More information

Impurity Identification using a Quadrupole - Time of Flight Mass Spectrometer QTOF

Impurity Identification using a Quadrupole - Time of Flight Mass Spectrometer QTOF Impurity Identification using a Quadrupole - Time of Flight Mass Spectrometer QTOF PUSHER TOF DETECTOR ZSPRAY TM Ion Source SAMPLING CONE SKIMMER RF HEXAPOLE RF HEXAPOLE QUADRUPOLE IN NARROW BANDPASS MODE

More information

LC/MS/MS SOLUTIONS FOR LIPIDOMICS. Biomarker and Omics Solutions FOR DISCOVERY AND TARGETED LIPIDOMICS

LC/MS/MS SOLUTIONS FOR LIPIDOMICS. Biomarker and Omics Solutions FOR DISCOVERY AND TARGETED LIPIDOMICS LC/MS/MS SOLUTIONS FOR LIPIDOMICS Biomarker and Omics Solutions FOR DISCOVERY AND TARGETED LIPIDOMICS Lipids play a key role in many biological processes, such as the formation of cell membranes and signaling

More information

Targeted and untargeted metabolic profiling by incorporating scanning FAIMS into LC-MS. Kayleigh Arthur

Targeted and untargeted metabolic profiling by incorporating scanning FAIMS into LC-MS. Kayleigh Arthur Targeted and untargeted metabolic profiling by incorporating scanning FAIMS into LC-MS Kayleigh Arthur K.Arthur@lboro.ac.uk Introduction LC-MS is a highly used technique for untargeted profiling analyses

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

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

New Frontiers for MS in Metabolipidomics

New Frontiers for MS in Metabolipidomics New Frontiers for MS in Metabolipidomics Giuseppe Astarita, PhD Principal Scientist Discovery & Life Sciences Milford, MA USA 2011 Waters Corporation 1 Why Metabolipidomics? Biomedical Sciences o Biomarker

More information

LC-MS/MS quantitative analysis of Polyunsaturated Omega 3, 6,7 and 9 Fatty Acids in Serum for

LC-MS/MS quantitative analysis of Polyunsaturated Omega 3, 6,7 and 9 Fatty Acids in Serum for POSTER NOTE 64921 LC-MS/MS quantitative analysis of Polyunsaturated Omega 3, 6,7 and 9 Fatty Acids in Serum for Research Use LC-MS/MS quantitative analysis of Poly Rory M Doyle*, Thermo Scientific, Inc,

More information

Essential Lipidomics Experiments using the LTQ Orbitrap Hybrid Mass Spectrometer

Essential Lipidomics Experiments using the LTQ Orbitrap Hybrid Mass Spectrometer Application Note: 367 Essential Lipidomics Experiments using the LTQ rbitrap Hybrid Mass Spectrometer Thomas Moehring 1, Michaela Scigelova 2, Christer S. Ejsing 3, Dominik Schwudke 3, Andrej Shevchenko

More information

Methods in Mass Spectrometry. Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University

Methods in Mass Spectrometry. Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University Methods in Mass Spectrometry Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University Sample Engineering Chemistry Biology Life Science Medicine Industry IDF / Police Sample

More information

Supporting Information

Supporting Information Supporting Information Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry Qiuhui Xuan 1,2#, Chunxiu Hu 1#, Di Yu 1,2,

More information

Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS

Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS Alexei Belenky CASSS, Meritage Resort, Napa 2008 Outline Role of lipid analysis in drug discovery and disease diagnostics Analytical tools and

More information

Analysis of HMF by HPLC

Analysis of HMF by HPLC COST Action 927 Training School Building Skills on the Analysis of Thermal Process Contaminants in Foods 22-26 October 2007, Ankara Analysis of HMF by HPLC Vural Gökmen O O OH Background O COOH O R 2 Carbonyl

More information

4-Plex itraq Based Quantitative Proteomic Analysis Using an Agilent Accurate -Mass Q-TOF

4-Plex itraq Based Quantitative Proteomic Analysis Using an Agilent Accurate -Mass Q-TOF 4-Plex itraq Based Quantitative Proteomic Analysis Using an Agilent Accurate -Mass Q-TOF Application Note Authors H. C. Harsha, G. S. S. Kumar, and A. Pandey Institute of Bioinformatics Bangalore India

More information

Ultra Performance Liquid Chromatography Coupled to Orthogonal Quadrupole TOF MS(MS) for Metabolite Identification

Ultra Performance Liquid Chromatography Coupled to Orthogonal Quadrupole TOF MS(MS) for Metabolite Identification 22 SEPARATION SCIENCE REDEFINED MAY 2005 Ultra Performance Liquid Chromatography Coupled to Orthogonal Quadrupole TOF MS(MS) for Metabolite Identification In the drug discovery process the detection and

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

MASS SPECTROMETRY IN METABOLOMICS

MASS SPECTROMETRY IN METABOLOMICS For personal use only. Please do not reuse or reproduce without the author s permission MASS SPECTRMETRY IN METABLMICS Pavel Aronov Stanford Mass Spectrometry Users Meeting August 21, 2008 rigin of Metabolomics

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

Mass spectra of peptides and proteins - and LC analysis of proteomes Stephen Barnes, PhD

Mass spectra of peptides and proteins - and LC analysis of proteomes Stephen Barnes, PhD Mass spectra of peptides and proteins - and LC analysis of proteomes Stephen Barnes, PhD 4-7117 sbarnes@uab.edu Overview A mass spectrum Electrospray MS Analysis of intact proteins Molecular weight calculations

More information

Characterization of an Unknown Compound Using the LTQ Orbitrap

Characterization of an Unknown Compound Using the LTQ Orbitrap Characterization of an Unknown Compound Using the LTQ rbitrap Donald Daley, Russell Scammell, Argenta Discovery Limited, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex, CM19 5TR, UK bjectives unknown

More information