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)
Proteomics: the main goals Protein Localization Quantification Identification Proteomics Structure Modifications Function Activity
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.
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
Several enzymes, each one with specific sites of hydrolysis, are currently available for protein digestion: example
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)
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
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).
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.
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):
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).
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.
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
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
MALDI-ToF-MS: relevant information for proteomics Integral protein mixture 1607.06 Tryptic peptide mixture 748.09 1884.32 1378.23 1502.02 1271.10 1815.13 11500 12000 12500 13000 13500 Mass (m/z) 600 800 1000 1200 1400 1600 1800 2000 2200 Mass (m/z) Molecular weight determination (up to 500000 Da) Competition for ionization between proteins Difficult quantification Peptide Mass Fingerprinting (PMF) Competition for ionization between peptides Difficult quantification Difficult coupling with separation techniques
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.
LC-ESI-MS: separation and analysis of integral proteins Relative Abundance 100 90 80 70 60 50 40 30 20 Pasteurized milk MS-TIC 1.67 -La 8.96 -Lg-B 11.64 -Lg-A 12.62 Relative abundance 100 80 60 40 20 -LgB MW = 18276 1143.3 +17 +16 +15 1075.9 1219.5 +14 +18 1306.6 1016.5 +13 1407.1 +12 +19 962.9 1524.0 +11 +10 1662.3 1828.5 10 0 0 2 4 6 8 10 12 14 16 Time / min 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z Relative abundance 100 80 60 40 20 0 -La MW = 14178 1013.7 1182.5 1290.1 1091.7 +14 +13 +12 +11 +10 1419.0 1576.5 1773.6 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z +9 +8 Relative abundance 100 1225.2 -LgA 80 1148.7 MW = 18363 +17 +14 60 1081.2 1312.3 +12 +11 40 +18 +13 1531.1 1670.3 1021.3 1413.5 20 +19 +10 1837.3 967.5 0 +16 +15 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z I. Losito, T. Carbonara, L. Monaci, F. Palmisano Anal. Bioanal. Chem. 389, 2007, 2065
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 = 10000-20000) 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
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 = 3530.33 Da [M+H] + (m/z) = 0.168 corrente Th ionica su m/z 467.288 Even long tryptic peptides can be detected, due to recognition of high charge states by high resolution MS.
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.
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.
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.
Several candidate proteins are usually listed as output:
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=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded 190 180 170 160 150 140 130 120 110 100 90 80 70 60 847.59 869.07 927.59 1022.56 1440.00 PMF 1479.98 1567.94 1640.16 Max. 1305.0 counts. 50 1305.87 1163.77 1481.98 40 1296.86 30 1249.77 871.07 1050.55 20 789.53 1024.56 1283.91 1417.93 1142.86 1443.01 857.14 1073.03 1386.76 1595.95 1824.09 10 978.60 1108.71 1292.95 1501.84 1616.921790.10 0 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z, amu 847.5896 869.0722 922.5712 923.5815 927.5904 1022.5551 1050.5533 1163.7695 1164.7531 1193.7393 1249.7705 1250.8103 1296.8556 1297.8499 1305.8668 1416.8929 1440.0008 1479.9773 1482.9583 1567.9417 1640.1635 1824.06 Best candidate database search
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.
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) 100 90 80 735.1 m/z = 1052.5 Relative abundance 70 60 50 40 30 20 10 0 887.3 806.2 491.1 1052.5 788.5 570.3 562.2 471.2 642.4 318.0 300 350 450 650 400 500 550 600 700 750 800 850 900 950 1000 1050 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.
LC-ESI-MS/MS, MS n analysis of peptides: fundamental stages Full scan MS acquisition (50-2000 Th) Processing of MS spectra for each peak Ion current extraction on specific m/z ratios MS/MS (MS 3 ) acquisitions Relative Abundance 100 90 80 70 60 50 40 30 20 Full scan MS chromatogram (base peak) 38.49 38.91 24.38 39.40 36.46 47.15 29.24 47.82 44.56 31.68 35.48 40.30 4.78 10 0 0 5 10 15 20 25 30 35 40 45 Retention time / min Relative Abundance Relative Abundance Relative Abundance 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 100 80 60 40 20 0 100 80 60 38.46 m/z = 1882.1 38.21 m/z = 1052.5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 38.42 m/z = 292.0 40 20 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 Retention time / min 100 90 t r = 38.49 min 1882.1 100 90 735.1 m/z = 1052.5 Relative Abundance 80 70 60 50 40 30 20 10 0 941.5 292.0 421.1 1052.5 731.4 1151.5 1701.0 181.1 617.1 826.5 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z Relative abundance 80 70 60 50 40 30 20 10 0 887.3 491.1 806.2 570.3 1052.5 562.2 788.5 318.0 471.2 642.4 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 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
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.
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.
Peptide bond: the key of peptide fragmentation pk 1 ~ 2.2 pk 2 ~ 9.4 pk R
The 20 proteinogenic aminoacids: main properties
MS-relevant information on the 20 proteinogenic aminoacids
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:
Alternatively, the positive charge can be located on a fragment containing the aminic end: Resonance stabilization
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:
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.
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
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 1 147 Arg y 1 175 b 1 ions are almost never observed in CID/CAD MS/MS spectra
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:
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).
An example of MS/MS spectrum interpretation: [Glu] 1 fibrinopeptide B MW 1569.669 [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
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.
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 114-117 is due to preliminary peptide derivatization with the itraq TM reagents, developed by Applied Biosystems for relative/absolute protein quantification.
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.
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.
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):
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
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 5 10-3 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.
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.
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
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:
MS and MS/MS analysis, along with appropriate extraction/separation techniques, play a key role in identifying phosphorylation sites:
Graphic reconstruction of a 2D-PAGE gel relevant to brain proteins (real dimensions: 10 14 cm)
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.
The MS/MS spectrum obtained on the digestive peptide enables an identification of the corresponding sequence, showing the phosphorylation site:
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.
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.
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:
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.
In this example, SELDI-MS is used to evaluate the kinetics of dephosphorylation of peptide ISpYGRKKRRQRRRP using alkaline phosphatase:
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
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.
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
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.
extracellular matrix Cholesterol cytoplasma Glycero-phospholipids Glycolipids
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 22 11 6 14 0 7 0 0 12 Erythrocyte 24 31 8,5 15 2,2 7 0 0 3 Hepatocyte 30 18 14 11 4 9 0 0 0 Mitochondr. (Internal) 3 45 2,5 24 6 1 2 18 0 E. coli 0 0 0 80 0 0 15 5 0 PG = phosphatidyl-glycerol, CL = cardiolypines
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
Palmitic (16:0) Stearic (18:0) Oleic (18:1) Linoleic (18:2) Linolenic (18:3) Arachidonic (20:4) DHA* (22:6) Others 80 70 60 50 40 30 20 10 0 Incidence of unsaturated fatty acids in PL Human erythrocites Egg Bovine brain fatty acids DHA = DocosaHexaenoic Acid % in total fatty acids
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.
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) 911-917
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
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) 311-314
Shotgun lipidomics: ESI-MS Acyl-carnitines GalC: GalactoCerebrosides FFA: Free Fatty Acids X. Han, R.W. Gross J. Lip. Res. 44 (2003) 1071-1079
Shotgun lipidomics: ESI-MS X. Han, R.W. Gross J. Lip. Res. 44 (2003) 1071-1079
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) 191-198
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) 246-254
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) 285-289
RPC separation of complex lipid mixtures 100 80 60 Relative Abundance 40 20 0 100 80 PC(16:0/18:2) 69.69 PC(16:0/18:1) 76.20 60 40 20 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 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
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) 231-247
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) 231-247
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
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) 6405-6413
extracted Ion Current (XIC) chromatograms 100 80 60 40 20 0 100 1.82 2.21 1.24 4.84 13.22 15.19 17.51 TIC ESI(+) Relative Abundance 80 60 40 20 0 100 80 60 40 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) 20 0 100 80 60 40 20 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) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min) I. Losito et al., Anal. Chem. 85 (2013) 6405-6413