Atmospheric Pressure Matrix-assisted Laser Desorption/ionization Research Articles

Development and Characterization of a Field-Deployable Ion-Trap Mass Spectrometer with an Atmospheric Pressure Interface

A field-deployable quadrupole ion-trap mass spectrometer with an atmospheric pressure interface is designed, built, and characterized. The instrument enclosure (48 cm × 43 cm × 42 cm) includes a roughing pump and a helium lecture bottle; the total weight of the instrument is 68 kg. Peak power consumption during the instrument operation is ∼500 W. The instrument has a mass range of m/z 30-2500, across which it provides better than unit mass resolution. The typical peak width at half height is 0.3 Th for a scan speed of 4000 Th/s. Operation of the instrument with electrospray and atmospheric-pressure matrix-assisted laser desorption ionization (AP-MALDI) ion sources is demonstrated. AP-MALDI analysis of low femtomole amounts of peptides reveals that the sensitivity of the instrument is on par with modern commercially available quadrupole ion-trap mass spectrometers. Tandem mass spectrometry capabilities of the instrument include simultaneous isolation and fragmentation of several different compounds. Two ways to reduce the size, weight, and power consumption of the portable instrument were explored, and results of these initial studies are presented. One of the ways includes utilization of hydrogen as a buffer gas for operation of the ion-trap mass analyzer in combination with a metal hydride method for storage of hydrogen in a compact rechargeable cartridge. Furthermore, careful selection of the inlet capillary dimensions allowed to eliminate the first "1 Torr" stage of the differential pumping without any significant loss of the instrument sensitivity. The elimination of this first pumping stage removed two turbo drag pumps, which substantially decreased the instrument's maximum power consumption (to ∼300 W in peak use, and ∼150 W during standby), as well as its size (to 30 cm × 43 cm × 50 cm) and weight (to 35 kg).

Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e., N-terminus and ε-NH(2) of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a noncovalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas-phase ion/ion reactions involving MALDI-derived ions. In this case, covalent and electrostatic modification charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides.

Single Cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100,000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.

Probing the mechanisms of ambient ionization by laser‐induced fluorescence spectroscopy

The ionization mechanisms of several atmospheric pressure ion sources based on desorption and ionization of samples deposited on a surface were studied. Home-built desorption electrospray ionization (DESI), laserspray ionization (LSI), and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) sources were characterized using low-molecular-weight compounds, in particular fluorescent dyes. Detection of the desorbed and ionized species was performed by laser-induced fluorescence and ion cyclotron resonance mass spectrometry. The dependences of the signal intensities on various experimental parameters were studied. The data obtained reveals common features, such as formation of solvated species and clusters in the ionization processes, in all of the techniques considered.

Atmospheric pressure visible-wavelength MALDI-MS

An atmospheric pressure (AP) visible-wavelength MALDI source was developed and coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer. This instrument employed a pulsed laser emitting light at a wavelength of 532nm to desorb and ionize samples such as dyes, oligosaccharides, peptides, and synthetic polymers at atmospheric pressure. Ions formed were analyzed by the Q-TOF in both MS and tandem MS (MS/MS) modes. Several visible-wavelength absorbing dyes were successfully utilized and studied as matrices. Among these dyes, nuclear fast red (NFR), rhodamine B isothiocyanate (RITC), and 5(6)-carboxytetramethyl-rhodamine N-succinimidyl ester (TAMRA, SE) are reported as effective visible-wavelength MALDI-MS matrices for the first time. This report demonstrates applicability of visible-wavelength AP MALDI-MS and AP MALDI MS/MS to the detection and structural analysis of dye molecules, biomolecules, and synthetic polymers.

Structural Characterization of Monosialo-, Disialo- and Trisialo-gangliosides by Negative Ion AP-MALDI-QIT-TOF Mass Spectrometry with MSn Switching

The atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) is a quite convenient soft ionization for biomolecules, keeping analytes atmospheric conditions instead of high vacuum conditions. In this study, an AP-MALDI ion source has been coupled to a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer, which is able to perform MS(n) analysis. We applied this system to the structural characterization of monosialogangliosides, GM1 (NeuAc) and GM2 (NeuAc), disialogangliosides, GD2 (NeuAc, NeuAc), GD1a (NeuAc, NeuAc) and GD1b (NeuAc, NeuAc) and trisialoganglioside GT1a (NeuAc, NeuAc, NeuAc). In this system, the negative ion mass spectra of MS, MS(2) and MS(3), a set of three mass spectra, were able to measure within 2s per cycle. Thus, obtained results demonstrate that the negative ion mode MS, MS(2) and MS(3) spectra provided sufficient information for the determination of molecular weights, oligosaccharide sequences and ceramide structures, and indicate that the AP-MALDI-QIT-TOF mass spectrometry keeping analytes atmospheric conditions with MS(n) switching is quite useful and convenient for structural analyses of various types of sialic acid-containing GSLs, gangliosides.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API–MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API–MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at −80 °C for 18 months or at −20 °C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API–MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.

AP-MALDI imaging of neuropeptides in mouse pituitary gland with 5 μm spatial resolution and high mass accuracy

MALDI MS imaging is a powerful tool to visualize the spatial distribution of endogenous biomolecules such as lipids or neuropeptides. Direct identification of analytes is often difficult due to the complexity of biological tissue samples. Today reliable analyte identification is routinely done with mass spectrometers featuring high mass resolving power, high mass accuracy and MS/MS capability. These mass spectrometers, however, typically have a rather poor spatial resolution when used in MALDI MS imaging experiments. In this work a linear ion trap orbital trapping mass spectrometer combined with an in-house developed atmospheric pressure MALDI imaging ion source was used to image neuropeptides in mouse pituitary gland with a spatial resolution of 5 μm. Ten neuropeptides were identified by their accurate mass in the mass range up to 2500 u via targeted database search. The ion images of the peptides show down to the cellular level that their appearances within the pituitary gland are restricted to accurately defined tissue types. This is in excellent agreement with the gland's structure and biological function. The identity of the neuropeptides was confirmed by additional MS/MS measurements from single 10 μm sample spots, obtained directly from tissue. Furthermore, MS/MS imaging of two different peptides at 10 μm spatial resolution resulted in product ion images, which were in good accordance with the distributions of their peptide precursor ions, confirming their identity and excluding possible analyte interferences. Since the method offers high performance mass spectrometry in combination with high spatial resolution, it appears to be ideally suited for imaging peptide signatures on a cellular level with high confidence in identification and with high sensitivity.

Laserspray Ionization, a New Atmospheric Pressure MALDI Method for Producing Highly Charged Gas-phase Ions of Peptides and Proteins Directly from Solid Solutions

The first example of a matrix-assisted laser desorption/ionization (MALDI) process producing multiply charged mass spectra nearly identical to those observed with electrospray ionization (ESI) is presented. MALDI is noted for its ability to produce singly charged ions, but in the experiments described here multiply charged ions are produced by laser ablation of analyte incorporated into a common MALDI matrix, 2,5-dihydroxybenzoic acid, using standard solvent-based sample preparation protocols. Laser ablation is known to produce matrix clusters in MALDI provided a threshold energy is achieved. We propose that these clusters (liquid droplets) are highly charged, and under conditions that produce sufficient matrix evaporation, ions are field-evaporated from the droplets similarly to ESI. Because of the multiple charging, advanced mass spectrometers with limited mass-to-charge range can be used for protein characterization. Thus, using an Orbitrap mass spectrometer, low femtomole quantities of proteins produce full-range mass spectra at 100,000 mass resolution with <5-ppm mass accuracy and with 1-s acquisition. Furthermore, the first example of protein fragmentation using electron transfer dissociation with MALDI is presented.

Immunoglobulin G Glycopeptide Profiling by Matrix-Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 microL of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples.

Imaging Mass Spectrometry Unravels the Mystery of Life

Imaging mass spectrometry (IMS) visualizes the distribution of a wide range of biomolecules in cells and tissue sections. IMS can analyze a variety of biomolecules including unexpected metabolites, without labels in a non-targeted way. We developed AP-MALDI (atmospheric pressure matrix assisted laser desorption/ionization) which is equipped with an atmospheric pressure ion-source chamber. It allows us to analyze flesh samples with minimal loss of intrinsic water or volatile compounds.

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI- and AP-MALDI-ITMS, forming various types/species of molecular ions (e.g. [M]+*, [M+H]+, [M+Na]+ or [M-2H+H]+). A few compounds could be analyzed by negative ion ESI-MS, too, but none by negative ion AP-MALDI-MS. The influence of target materials in AP-MALDI-MS (gold- and titanium nitride (TiN)-covered stainless steel, micro-diamond-covered hard metal, hand-polished and sand-blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP-MALDI-MS but in general smooth surfaces were superior to rough surfaces. Finally the gold-covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Method for Selectively Producing Either Singly or Multiply Charged Molecular Ions

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is noted for its ability to produce primarily singly charged ions. This is an attribute when using direct ionization for complex mixtures such as protein digests or synthetic polymers. However, the ability to produce multiply charged ions, as with electrospray ionization (ESI), has advantages such as extending the mass range on mass spectrometers with limited mass-to-charge (m/z) range and enhancing fragmentation for structural characterization. We designed and fabricated a novel field free transmission geometry atmopsheric pressure (AP) MALDI source mounted to a high-mass resolution Orbitrap Exactive mass spectrometer. We report the ability to produce at will either singly charged ions or highly charged ions using a MALDI process by simply changing the matrix or the matrix preparation conditions. Mass spectra with multiply charged ions very similar to those obtained with ESI of proteins such as cytochrome c and ubiquitin are obtained with low femtomole amounts applied to the MALDI target plate and for peptides such as angiotensin I and II with application of attomole amounts. Single scan acquisitions produce sufficient ion current even from proteins.

On The Time Scale of Internal Energy Relaxation of AP-MALDI and nano-ESI Ions in a Quadrupole Ion Trap

Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results. Collision induced dissociation (CID) has been used to probe various properties of ions contained in a QITMS. It is shown experimentally that when trapping large numbers of ions, an effective dc trapping voltage is induced that varies with changes in the size of the ion cloud. A decrease in the resonant frequency for maximum CID efficiency is observed as the cool time between parent ion isolation and CID is increased. Ion trajectories in a QITMS are simulated to demonstrate how ion density changes over the course of parent ion isolation. The effect of space charge on ion motion is simulated, and Fourier transformations of ion axial motion plus simple calculations corroborate the experimentally observed transient frequency shifts. The relative stability of ions formed by AP-MALDI and nano-ESI is compared under low charge density conditions. These data show that the ions have reached equilibrium internal energy and, thus, that differences in dissociation onsets and "50% fragmentation efficiency points" between the ionization mechanisms are due to the formation of distinct ion conformations as previously shown in reference [28]. The conclusions of Konn et al. [14] are based on invalid experimental procedures as well as inappropriate comparisons of QITMS data to low-pressure FT-ICR data.

MALDI, AP/MALDI and ESI techniques for the MS detection of amyloid β-peptides

Amyloid β-peptides (Aβs) are involved in several neuropathological conditions such as Alzheimer's disease and considerable experimental evidences have emerged indicating that different proteases play a major role in regulating the accumulation of Aβs in the brain. Particularly, insulin-degrading enzyme (IDE) has been shown to degrade Aβs at different cleavage sites, but the experimental results reported in the literature and obtained by mass spectrometry methods are somehow fragmentary. The detection of Aβs is often complicated by solubility issues, oxidation artifacts and spontaneous aggregation/cleavage and, in order to rationalize the different reported results, we analyzed Aβs solutions by three different MS approaches: matrix assisted laser desorption ionization-time of flight (MALDI-TOF), atmospheric pressure (AP) MALDI ion trap and electrospray ionization (ESI) ion trap. Differences in the obtained results are discussed and ESI is chosen as the most suitable MS method for Aβs detection. Finally, cleavage sites produced by interaction of Aβs with IDE are identified, two of which had never been reported in the literature.

On-chip solid-phase extraction pre-concentration/focusing substrates coupled to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP-MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP-MALDI suffers from less-than-optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal-to-noise ratio (S/N) gains observed when an on-chip dynamic pre-concentration/focusing approach is coupled to AP-MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP-MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200-900 for hydrophobic substrates, and 150-400 for weak cation-exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/microL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter-day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP-MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads.

Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme

Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.

Modified Silver Nanoparticle as a Hydrophobic Affinity Probe for Analysis of Peptides and Proteins in Biological Samples by Using Liquid−Liquid Microextraction Coupled to AP-MALDI-Ion Trap and MALDI-TOF Mass Spectrometry

A new approach of using modified silver nanoparticles (AgNPs) in toluene as hydrophobic affinity probes for the separation and preconcentration of peptides and proteins in biological samples prior to atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry and matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry has been successfully demonstrated. To our best knowledge, for the first time, the modified AgNPs with hydrophobic ligands, such as dodecanethiol (DT) and octadecanethiol (OT) in toluene, were used for the liquid-liquid microextraction (LLME) of peptides and proteins through the hydrophobic interactions. In the present investigation, gramicidin was chosen as a model compound to assess the hydrophobic extraction with the modified AgNPs. The optimum extraction efficiency of gramicidine was observed at pH 7.0 for 1.5 h of extraction time with 7% addition of salt. Compared to the conventional use of AP-MALDI-MS, a 266-388-fold improvement in the limit of detection (LOD) for gramicidin was obtained in urine and plasma samples. The lowest concentration of gramicidin that was detected by using modified AgNPs in urine and plasma samples was 0.13 and 0.16 microM, respectively. Furthermore, the proposed method was demonstrated for the extraction of other long chain proteins, like myoglobin, ubiquitin, and bovine serum albumin, in a sample solution by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The major feature of the newly synthesized modified AgNPs was that the target species could be efficiently separated and preconcentrated without sample loss prior to MALDI-MS detection for the sensitive and effective analysis of peptides and proteins in biological samples.