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Analytica Chimica Acta (v.627, #1)
Recent applications of capillary electrophoresis–mass spectrometry (CE–MS): CE performing functions beyond separation by Chandra A. Nesbitt; Haixia Zhang; Ken K.-C. Yeung (pp. 3-24).
Capillary electrophoresis is one of the separation tools commonly used in conjugation with mass spectrometry. Its primary purpose is to resolve the components in a sample mixture prior to mass spectral identification. Moreover, an increasing number of applications reported in the literature involve the use of CE for additional purposes, such as sample preparation and derivatization, and the study of biochemical properties. This review provides an overview on the various roles of CE beyond that of a simple separation tool. While the scope focuses on the area of interest rather than a predefined time period, the majority of the references highlighted were initially published within the past five years.
Keywords: Sample enrichment; Low-volume fractionation; In-capillary reactions; In-capillary kinetic measurements
Twenty years of interface development for capillary electrophoresis–electrospray ionization–mass spectrometry by E. Jane Maxwell; David D.Y. Chen (pp. 25-33).
Capillary electrophoresis–electrospray ionization–mass spectrometry has the potential to become a preferred tool for the analysis of biological mixtures and other complex samples. The development of improved interfaces in the past twenty years has been critical in demonstrating the feasibility of this technique. However, a compromise still exists between interfaces that give optimal performance and those that are practical for commercial applications. The first section of this review focuses on the technological advances in CE–ESI–MS as they relate to the key interface features for both sheath-flow and sheathless systems: delivery of the sheath liquid, shaping of the emitter tip, formation of electrical contact, and practicality in terms of ease of use and lifetime. In the second section, we review the fundamental processes that affect interface performance. Because of the complex natures of both capillary electrophoresis and electrospray ionization, flow rate, arrangement of the electrical circuit, electrochemistry, tip geometry and location of electrical contact must all be carefully managed in the design of a successful interface.
Keywords: Capillary electrophoresis; Electrospray ionization; Mass spectrometry; Interface development
State-of-the-art in atmospheric pressure photoionization for LC/MS by Damon B. Robb; Michael W. Blades (pp. 34-49).
This review presents our perspective on the state-of-the-art of atmospheric pressure photoionization (APPI) for LC/MS. Its focus is on APPI's capabilities and how to utilize them fully. The introduction includes a brief recounting of the history of APPI's development, as well as a summary of its operating principles and current position in the field. The primary ionization mechanisms in APPI are then addressed, including direct analyte photoionization (PI), dopant/solvent PI, and thermospray. Next a summary of the ion–molecule reaction pathways available for analyte ionization is presented, along with the conditions required for activating them. APPI's performance characteristics are then examined. In effect, this review is an interim report on progress made since Rafaelli and Saba concluded that “The ability…to direct the preferential ion formation towards one particular type…can be extremely useful for qualitative and quantitative determinations. For this purpose, a better insight in the processes involved in the ionization step is strongly needed” [A. Raffaelli, A. Saba, Mass Spectrom. Rev. 22 (2003) 318]. In the conclusion, we focus on areas of APPI technology identified as being either unoptimized or largely unexplored, and having the potential to be improved upon—the crux being that with further research and development improvements in the performance, capabilities, and ease-of-use of APPI may reasonably be anticipated.
Keywords: Atmospheric pressure photoionization; Liquid chromatography/mass spectrometry; Review
Current approaches for global post-translational modification discovery and mass spectrometric analysis by Michael D. Hoffman; Matthew J. Sniatynski; Juergen Kast (pp. 50-61).
More and more attention is being focused on the analysis of post-translational modifications (PTMs) on proteins as researchers are continually learning how essential they are for proper cellular function. As there are hundreds of different types of known PTMs, traditional methods of modification analysis are incapable of comprehensively monitoring for post-translational modifications, a task which is a necessity for truly understanding a cell's biology. This review highlights recent developments in novel multiplexed methods of PTM analysis including: fluorescent stain and immuno-based methods, hardware-based mass spectrometric methods and computational-based mass spectrometric methods. Many of these techniques show great promise and will likely be a valuable resource for the biological community.
Keywords: Post-translational modifications; Mass spectrometry; Marker ion; Neutral loss; Computational; Protein array
Recent developments in tandem mass spectrometry for lipidomic analysis by Nicole Zehethofer; Devanand M. Pinto (pp. 62-70).
This review will focus on the role of mass spectrometry in the emerging field of lipidomics. Particular emphasis will be placed on recent developments in the use of tandem mass spectrometry methods in lipid analysis using low-energy collision induced dissociation (CID). After a brief discussion on ionization techniques, novel ion-activation methods that allow for increased sensitivity and selectivity will be critically discussed. Examples will be drawn from the analysis of higher order lipids, specifically triacylglycerols (TAGs) and glycerophospholipids, as the numerous positional isomers and head groups present in these classes of lipids continue to pose a significant analytical challenge to the field of lipidomics. The role of bioinformatics in the development of lipidomics will also be discussed.
Keywords: Lipidomics; Diacylglycerols; Triacylglycerols; Tandem mass spectrometry; Ion activation
Interferences and contaminants encountered in modern mass spectrometry by Bernd O. Keller; Jie Sui; Alex B. Young; Randy M. Whittal (pp. 71-81).
With the invention of electrospray ionization and matrix-assisted laser desorption/ionization, scientists employing modern mass spectrometry naturally face new challenges with respect to background interferences and contaminants that might not play a significant role in traditional or other analytical techniques. Efforts to continuously minimize sample volumes and measurable concentrations increase the need to understand where these interferences come from, how they can be identified, and if they can be eliminated. Knowledge of identity enables their use as internal calibrants for accurate mass measurements. This review/tutorial summarizes current literature on reported contaminants and introduces a number of novel interferences that have been observed and identified in our laboratories over the past decade. These include both compounds of proteinaceous and non-proteinaceous nature. In the supplemental data a spreadsheet is provided that contains a searchable ion list of all compounds identified to date.
Keywords: Background ions; Interferences; Contaminants; Mass spectrometry; Matrix-assisted laser desorption/ionization; Electrospray ionization
Liquid chromatography electrospray ionization and matrix-assisted laser desorption ionization tandem mass spectrometry for the analysis of lipid raft proteome of monocytes by Nan Zhang; Andrew R.E. Shaw; Nan Li; Rui Chen; Allan Mak; Xiuying Hu; Nelson Young; David Wishart; Liang Li (pp. 82-90).
Lipid rafts are dynamic assemblies of cholesterol and glycolipid that form detergent-insoluble microdomains within membrane lipid bilayers. Because rafts can be separated by flotation on sucrose gradients, interrogation by mass spectrometry (MS) provides a valuable new insight into lipid raft function. Here we combine liquid chromatography (LC) electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) MS/MS to corroborate and extend our previous description of lipid raft proteomes derived from the monocytic cell line THP-1. Interestingly, LC-ESI and MALDI MS/MS identify largely non-overlapping, and therefore, potentially complementary protein populations. Using the combined approach, we detected 277 proteins compared to 52 proteins obtained with the original gel-based MALDI MS. We confirmed the presence of 47 of the original 52 proteins demonstrating the consistency of the lipid raft preparations. We demonstrated by immunoblotting that Rac 1 and Rac 2, two of the 52 proteins we failed to confirm, were indeed absent from the lipid raft fractions. The majority of new proteins were cytoskeletal proteins and their regulators, proteins implicated in membrane fusion and vesicular trafficking or signaling molecules. Our results therefore, confirm and extend previous evidence indicating lipid rafts of monocytic cells are specialized for cytoskeletal assembly and vesicle trafficking. Of particular interest, we detected SNAP-23, basigin, Glut-4 and pantophysin in lipid rafts. Since these proteins are implicated in both vesicular trafficking and gamete fusion, lipid rafts may play a common role in these processes. It is evident that the combination of LC-ESI and LC-MALDI MS/MS increases the proteome coverage which allows better understanding of the lipid raft function.
Keywords: Liquid chromatography–mass spectrometry; Proteomics; Lipid Raft; Membrane Proteins
Column-switching reversed phase–hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water by Feng Qin; Yuan Yuan Zhao; Michael B. Sawyer; Xing-Fang Li (pp. 91-98).
We report a column-switching liquid chromatography (LC) tandem mass spectrometry (MS/MS) method for highly sensitive determination of both free estrogens (estrone, estradiol, and estriol) and their conjugates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate, estrone-3-glucuronide, estradiol-3-glucuronide, estriol-16-glucuronide, and estriol-3-glucuronide) in river water. This technique combines reversed phase (RP) chromatographic separation of the dansyl chloride derivatized free estrogens and hydrophilic interaction liquid chromatographic (HILIC) separation of the estrogen conjugates with multiple reaction monitoring (MRM). Using this new method, sensitivity increases 100- to 1000-fold for free estrogens and 2- to 10-fold for estrogen conjugates over RPLC–MS/MS alone. Method detection limits (MDL) range from 0.038 to 6.9ngL−1 with accuracy of 68–105% and precision of 1.7–17%. We successfully used this method to analyze river water samples collected from the North Saskatchewan River at the same location and detected trace concentrations of estrone (0.042ngL−1) and estrone-3-sulfate (0.84ngL−1), demonstrating the application of this method for environmental analysis.
Keywords: Estrogens; Estrogen conjugates; Column-switching; Hydrophilic interaction liquid chromatography (HILIC); Tandem mass spectrometry
Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity by Roslyn Dillon; Michael J. Greig; B. Ganesh Bhat (pp. 99-104).
Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids—mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1−/−) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography–mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC50 of 247nM. The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases.
Keywords: Stearoyl-CoA desaturase; Oleic acid; Stearic acid; LCMS; Sterculic acid; Mass spectrometry
Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry by Patricia Soulard; Meg McLaughlin; Jessica Stevens; Brendan Connolly; Rocco Coli; Leyu Wang; Jennifer Moore; Ming-Shang T. Kuo; William A. LaMarr; Can C. Ozbal; B. Ganesh Bhat (pp. 105-111).
Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate ( Km=10.5μM). The assay was highly reproducible with an average Z′ value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC50 values of 0.88 and 0.12μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20μM – 14% conformation rate). Of the confirmed hits 172 had IC50 values of <10μM, including 111 <1μM and 48 <100nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.
Keywords: Abbreviations; SCD1; stearoyl coenzyme-A desaturase 1; HTS; high-throughput screening; CLA; conjugated linoleic acid; RF-MS; RapidFire Mass Spectrometry; TLC; thin-layer chromatographyStearoyl coenzyme-A desaturase 1; Fatty acid; Desaturase; High-throughput screening; Metabolic diseases; Lipid
Evaluation of high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of the mycotoxin zearalenone by Margaret McCooeye; Beata Kolakowski; Joe Boison; Zoltán Mester (pp. 112-116).
A high-field asymmetric waveform ion mobility spectrometry (FAIMS)-based method for the determination of the mycotoxin zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and β-zearalanol (β-ZAL), in a cornmeal (maize) matrix is described. Detection limits achieved using the FAIMS device coupled with electrospray ionization (ESI) and mass spectrometric (MS) detection are 0.4ngmL−1 for ZON and 3ngmL−1 for α-ZOL+β-ZOL, and β-ZAL. This represents a significant improvement when compared to detection limits determined using ESI-MS or ESI-tandem mass spectrometry (MSMS) analytical methods. The developed flow-injection (FIA)-ESI-FAIMS-MS method was applied to reference materials ERM-BC-716 and ERM-BC-717 certified for ZON and excellent agreement with the certified values was observed.
Keywords: Mass spectrometry; High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS); Mycotoxin zearalenone; Electrospray ionization
Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies by Qingrong Zhang; Elizabeth Crosland; Daniele Fabris (pp. 117-128).
The combination of chemical probing and high-resolution mass spectrometry constitutes a powerful alternative for the structural elucidation of biomolecules possessing unfavorable size, solubility, and flexibility. We have developed nested Arg-specific bifunctional crosslinkers to obtain complementary information to typical Cys- and Lys-specific reagents available on the market. The structures of 1,4-phenyl-diglyoxal (PDG) and 4,4′-biphenyl-diglyoxal (BDG) include two identical 1,2-dicarbonyl functions capable of reacting with the guanido group of Arg residues in proteins, as well as the base-pairing face of guanine in nucleic acids. The reactive functions are separated by modular spacers consisting of one or two benzene rings, which confer greater rigidity to the crosslinker structure than it is afforded by typical aliphatic spacers. Analysis by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown that the probes provide both mono- and bifunctional products with model protein substrates, which are stabilized by the formation of diester derivatives in the presence of borate buffer. The identification of crosslinked sites was accomplished by employing complementary proteolytic procedures and peptide mapping by ESI-FTICR. The results showed excellent correlation with the solvent accessibility and structural context of susceptible residues, and highlighted the significance of possible dynamic effects in determining the outcome of crosslinking reactions. The application of nested reagents with different spacing has provided a new tool for experimentally recognizing flexible regions that may be involved in prominent dynamics in solution. The development of new bifunctional crosslinkers with diverse target specificity and different bridging spans is expected to facilitate the structure elucidation of progressively larger biomolecular assemblies by increasing the number and diversity of spatial constraints available for triangulating the position of crosslinked structures in the three dimensions.
Keywords: Structural probing; Structure determination; Bifunctional crosslinking; Bis-(1,2-dicarbonyls); Arg-specific labels; ESI-FTICR mass spectrometry
ESI-MS characterization of a novel pyrrole–inosine nucleoside that interacts with guanine bases by Sarah E. Pierce; Courtney L. Sherman; Janarthanan Jayawickramarajah; Candace M. Lawrence; Jonathan L. Sessler; Jennifer S. Brodbelt (pp. 129-135).
Based on binding studies undertaken by electrospray ionization-mass spectrometry, a synthetic pyrrole–inosine nucleoside,1, capable of forming an extended three-point Hoogsteen-type hydrogen-bonding interaction with guanine, is shown to form specific complexes with two different quadruplex DNA structures [dTG4T]4 and d(T2G4)4 as well as guanine-rich duplex DNA. The binding interactions of two other analogs were evaluated in order to unravel the structural features that contribute to specific DNA recognition. The importance of the Hoogsteen interactions was confirmed through the absence of specific binding when the pyrrole NH hydrogen-bonding site was blocked or removed. While2, with a large blocking group, was not found to interact with virtually any form of DNA,3, with the pyrrole functionality missing, was found to interact non-specifically with several types of DNA. The specific binding of1 to guanine-rich DNA emphasizes the necessity of careful ligand design for specific sequence recognition.
Keywords: Electrospray ionization; Oligonucleotides; Non-covalent interactions; Hoogsteen binding
On the interaction of peptides with calcium ions as studied by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry: Towards peptide fishing using metal ion baits by Karl J. Jobst; Johan K. Terlouw; Theo M. Luider; Peter C. Burgers (pp. 136-147).
In a previous report [J.W. de Beukelaar, J.W. Gratama, P.A. Sillevis Smitt, G.M. Verjans, J. Kraan, Th.M. Luider, P.C. Burgers, Rapid Commun. Mass Spectrom. 21 (2007) 1282] on the quality assessment of synthetic peptides used in protein-spanning peptide pools by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) we noted that certain peptides showed remarkably intense signals for their calcium-containing analogues. Here we report on a detailed mass spectrometric study of the unimolecular chemistry of these calcium-containing peptides. By integration of the experimental findings with computational results derived from DFT and the CBS-QB3 model chemistry, we have traced the processes induced by Ca2+ attachment in the peptide ions.Key to our analysis is the observation that all of the studied calcium-bound peptides containing a threonine or serine residue show prominent losses of CH3CHO (from threonine) and/or CH2O (from serine) in both the positive and the negative ion mode. In the first step, Ca2+ attaches itself to a negatively charged in-chain carboxylate group. Next, electrophilic attack of the calcium ion on theCH(R)OH group of threonine (RCH3) or serine (RH) releases the hydroxyl proton which can then move to a suitable acceptor site, viz. a peptide bond. This leads to the formation of a very stable ionic bidentate structure. Upon collisional activation (MS/MS), this bidentate opens up leading to the loss of the exposed acetaldehyde or formaldehyde molecule, to yield another bidentate structure.MS/MS spectra of selected peptides interacting with other metal ions have also been investigated and it is found that only divalent ions follow the Ca2+-induced transformations.
Keywords: Fourier transform mass spectrometry; Calcium ion binding sites; Peptide binding; Bidentate structure; Ab initio calculations; Reaction mechanism
On the chemical resolution of the87Rb+ (s0)/87Sr+ (s1) isobaric interference: A kinetic search for an optimum reagent by Ping Cheng; Gregory K. Koyanagi; Diethard K. Bohme (pp. 148-153).
Room-temperature reactions of the atomic cations Sr+ and Rb+ have been surveyed systematically with a variety of gases using an Inductively-Coupled Plasma/Selected-Ion Flow Tube (ICP/SIFT) tandem mass spectrometer. Rate coefficients and product distributions have been measured in He buffer gas at 0.35Torr and 295K for reactions of Sr+ and Rb+ with CH3F, CH3Cl, N2O, CO2, CS2, SF6, D2O and NH3. Rb+ (s0) is seen to be quite inert with these molecules and reacts either slowly by molecule addition or not at all, while Sr+ (s1) is much more reactive with all these 8 molecules, especially with CH3F, CH3Cl, N2O and SF6. Sr+ reacts with CH3F and SF6 by F-atom transfer, with CH3Cl by Cl-atom transfer and with N2O by O-atom transfer, and the reaction rate coefficients are all quite high, k≥1.4×10−11cm3molecules−1s−1. The extreme differences in reactivity with CH3F, SF6, CH3Cl and N2O provide a chemical basis for the separation of isobaric interferences of87Rb+ and87Sr+ often encountered in ICP–MS. Among these four molecules, SF6 exhibits the largest difference in reactivity, almost a factor of 104, and so is identified as the kinetically recommended reagent for the chemical resolution of the isobaric interference of87Rb+ and87Sr+.
Keywords: Ion-molecular reactions; Chemical resolution; Rb/Sr; Isobaric interference
On-target digestion of collected bacteria for MALDI mass spectrometry by Alton J. Dugas Jr.; Kermit K. Murray (pp. 154-161).
An on-target protein digestion system was developed for the identification of microorganisms in collected bioaerosols using off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Bacteria analysis techniques based on MALDI-MS were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Bioaerosols were generated using a pneumatic nebulizer and infused into a chamber for sampling. An Andersen N6 single-stage impactor was used to collect the bioaerosols on a MALDI target. On-target digestion was carried out inside temporary mini-wells placed over the impacted samples. The wells served as miniature reactors for proteolysis. Collected test aerosol particles containing the protein cytochrome c and E. coli bacteria were proteolyzed in situ using trypsin or cyanogen bromide. A total of 19 unique proteins were identified for E. coli. Using the TOF-MS spectra of the digested samples, peptide mass mapping was performed using the MASCOT search engine and an iterative search technique.
Keywords: Matrix-assisted laser desorption/ionization; Bacteria; Digestion; Fingerprinting
Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry by Neerav D. Padliya; Troy D. Wood (pp. 162-168).
Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80°C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.
Keywords: Matrix-assisted laser desorption and ionization; Time-of-flight mass spectrometry; Peptide mass fingerprinting; Sample preparation; Sequence coverage