Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications

We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.     

Characterization of protein kinase A phosphorylation: multi-technique approach to phosphate mapping

A multi-technique approach to identification and mapping of phosphorylation on protein kinase A (PKA) is described. X-ray crystallography revealed phosphorylation at T197 and S338 while mass spectrometry (MS) on the intact protein suggested phosphorylation at three sites. Tryptic digestion, followed by MS, confirmed the presence of three phosphates. However, metal affinity treatment of the digest prior to MS revealed the presence of a fourth phosphopeptide. Subsequent analysis of the digests using liquid chromatography (LC) coupled with quadrupole ion trap (QIT) MS confirmed phosphorylation at S10 and S338 and suggested phosphorylation at S139 and T195/197. Unfortunately, identification of pS139 was inconclusive due to low signal intensity and early elution in reversed-phase LC while poor MS/MS data prevented localization of the phosphate to T195 or T197. Phosphopeptide modification with ethanethiol, followed by LC QIT-MS/MS, identified four phosphopeptides in a single experiment. In addition, the fragmentation data provided significantly more sequence information than data obtained from unmodified peptides. Data from this study suggested that PKA was completely phosphorylated at S10, T197, and S338 and partially phosphorylated at S139. These results illustrate that critical information can be lost unless multiple MS techniques are used for identification and validation of phosphorylation.     

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double- and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap

This report evaluates the use of a quadrupolar ion trap for quantitation in a bioanalytical laboratory. The evaluation was accomplished with the cross-validation of an LC–MS–MS quantitative method previously validated on a triple quadrupole mass spectrometer. The method was a multi-level determination of the anti-obesity drug, orlistat, in human plasma. The method has been refined previously on a triple quadrupole instrument to provide rapid sample throughput with robust reproducibility at sub-nanogram detection limits. Optimization of the method on the ion trap required improved chromatographic separation of orlistat from interfering plasma matrix components coextracted during the initial liquid–liquid extraction of plasma samples. The ion trap produces full-scan collision-induced dissociation mass spectra containing characteristic orlistat fragment ions that are useful for quantitation. Data collection on the ion trap required a precursor ion isolation width of 3.0 Da and optimal quantitative results were obtained when three fragment ions were monitored with a 1.8 Da window for each ion. Although a direct cross-validation between the ion trap and the tandem triple quadrupole mass spectrometer was not possible, quantitative results for orlistat comparable to those obtained from the triple quadrupole instrument were achieved by the ion trap with the modified method. The limit of quantitation for orlistat in plasma on the ion trap was 0.3 ng ml⁻¹ with a linear dynamic range of 0.3 to 10 ng ml⁻¹. Precision and accuracy varied from 4 to 15% over the quantitation range. The overall results provide an example of the utility of an ion trap in bioanalytical work.     

Mass‐Spectrometric Identification of Anandamide and 2‐Arachidonoylglycerol in Nematodes

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). In this study, AEA and 2‐AG were identified as endogenous products from nematodes by using electrospray‐ionization ion‐trap MS/MS (ESI‐IT‐MS) experiments operated in the positive‐ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2‐AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2‐AG were detected in the fat‐3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

Characterization of protein glycosylation using chip-based nanoelectrospray with precursor ion scanning quadrupole linear ion trap mass spectrometry.

Glycosylation is one of the most important posttranslational modifications affecting the functions of proteins and cell activities. Mass spectrometry (MS) has proven to be an effective tool for structural glycobiology and has helped gain an understanding of glycoprotein-mediated diseases. Although electro-spray ionization-tandem MS remains widely recognized as an effective means for oligosaccharide characterization, the hydrophilic nature of glycans has often caused the poor ionization efficiency requiring either derivatization or nanoelectrospray to improve detection sensitivity. In this report we describe the use of a chip-based infusion nanoelectrospray platform coupled with the hybrid triple quadrupole/linear ion trap for identification and characterization of glycosylation in complex mixtures. The high-mannose-type N-glycosylation in ribonuclease B was used to map the glycosylation site and obtain glycan structures. Using the chip-based nanoelectro-spray with precursor ion scanning linear ion trap MS, we were able to map the glycosylation site and obtain the glycan structures in ribonuclease B at 100 fmol/microL in a single analysis. In addition, a new, low-abundant glycoform with an additional hexose (Hex10GlcNAc2) attached to ribonuclease B was discovered. The results reported here demonstrate that the chip-based infusion nanoelectrospray ionization coupled to a quadrupole/linear ion trap platform is a valuable system, as it provides high sensitivity and stability for nanoelectrospray analysis, and allows extended acquisition time for completing precursor ion scanning and subsequent MS2 and MS3 information in a single analysis.     

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.     

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.     

Rapid protein identification using direct infusion nanoelectrospray ionization mass spectrometry

Current protein identification techniques are largely based on MALDI-TOF mass fingerprinting and LC-ESI MS/MS sequence tag analysis. Here we describe an improved method for rapid protein identification that uses direct infusion nanoelectrospray quadrupole time-of-flight (nanoESI QTOF) MS. Protein digests were analyzed without LC separation using nanoESI on a QSTAR XL MS/MS system in information dependent data acquisition mode. The protein identification conditions and parameters were extensively evaluated with in-solution and in-gel digested protein samples. Rapid identification of proteins was achieved and compared directly to the results obtained on the same samples using nanoflow HPLC-MS/MS on the QSTAR system. The increased throughput, reproducibility, the high data quality, and the ease of use make the direct infusion system an efficient and affordable technique for protein identification analysis.     

Confirmation of spectinomycin in milk using ion-pair solid-phase extraction and liquid chromatography–electrospray ion trap mass spectrometry

A confirmation procedure is described for residues of spectinomycin in bovine milk. Spectinomycin is extracted from raw milk using ion-pair reversed-phase solid-phase extraction. The extracts are ion-pair chromatographed on a polymeric reversed-phase column and analyzed on a quadrupole ion trap mass spectrometer equipped with an electrospray interface. MS–MS data are acquired in the scan mode of product ions deriving from m/z 333, the protonated molecular ion. The estimated limit of confirmation is between 0.05 and 0.1 μg/ml. The procedure was validated with control milk, fortified milk (0.1–5.0 μg/ml), and milk from cows dosed with spectinomycin.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.     

Ultra-fast quantitation of saquinavir in human plasma by matrix-assisted laser desorption/ionization and selected reaction monitoring mode detection

We present herein an ultra-fast quantitative assay for the quantitation of saquinavir in human plasma, without prior chromatographic separation, with matrix-assisted laser desorption/ionization using the selected reaction monitoring quantitation mode (MALDI-SRM/MS). The method was found to be linear from 5 to 10,000ng/ml using pentadeuterated saquinavir (SQV-d5) as an internal standard, and from 5 to 1000ng/ml using reserpine as internal standard (IS). Accuracy and precision were in the range of 101-108%, 3.9-11% with SQV-d5 and in the range 93-108%, 3.5-15% with reserpine. Plasma samples (250mul) were extracted with a mixture of ethyl acetate/hexane. MALDI spotting of the extract was automated using electrodeposition and the dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. A 96 spots MALDI plate was prepared within 20min in a fully unattended manner. Each sample was spotted four times and quantitation was based on the average of their analyte/IS area ratio. Samples were analyzed on a triple quadrupole linear ion trap (QqQ(LIT)) equipped with a high repetition laser source (1000Hz). The analysis time of one sample was approximately 6s, therefore 96 samples could be analyzed in less than 10min. With liquid-liquid extraction sample preparation no significant matrix effects were observed. Moreover, the assay showed sufficient selectivity for samples to be analyzed at the lower limit of quantification (LLOQ) in the presence of other antiretroviral drugs, without prior chromatographic steps. In parallel, to assess the selectivity of the assay with real samples, a liquid chromatography (LC)-SRM/MS method was developed and a cross validation with clinical samples was successfully performed.     

Full-scan high resolution accurate mass spectrometry (HRMS) in regulated bioanalysis: LC-HRMS for the quantitation of prednisone and prednisolone in human plasma

A liquid chromatography-full scan high resolution accurate mass spectrometry (LC-HRMS) method for quantifying prednisone and prednisolone in human plasma using a quadrupole time-of-flight mass spectrometer (Q-TOF) was developed. Plasma samples were extracted using a liquid-liquid extraction procedure. Full scan data were acquired in the TOF only mode and extracted ion chromatograms were generated post-acquisition with the exact masses of the analytes. The calibration range was 5-2500 ng/mL, with a Lower Limit of Quantitation (LLOQ) of 5 ng/mL. The assay accuracy was between 98.4% and 106.3%. The between-run (inter-day) and within-run (intra-day) precision were within 1.7% and 2.9%, respectively. The matrix effect was between 0.98 and 1.10 for the six different lots of human plasma evaluated. Pooled incurred samples were analyzed by the method and the results matched those obtained from an LC-MS/MS method. In addition, qualitative information on phospholipids, and other endogenous components were also extracted from the full-scan data acquired.     

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.     

Unequivocal Enantiomeric Identification and Analysis of 10 Chiral Pesticides in Fruit and Vegetables by QuEChERS Method Combined With Liquid Chromatography‐Quadruple/Linear Ion Trap Mass Spectrometry Determination

In this research, 10 chiral pesticides in fruits and vegetables were simultaneously determined using chiral liquid chromatography triple quadrupole‐linear ion trap hybrid mass spectrometry (LC‐QqLIT). The QuEChERS method was applied for sample preparation, and an enhanced product ion (EPI) scan was used to acquire tandem mass spectrometry (MS/MS) spectra for the library search. Parameters including limit of detection (LOD), limit of quantification (LOQ), linearity, relative standard deviation (RSD), and matrix effects were evaluated in five representative matrices (strawberry, leek, cowpea, tomato, and eggplant). Good linearity with coefficient of determination (r²) ≥0.997 was obtained for all 20 enantiomers in these five matrices over the range from 1.0 to 250 µg L^‐1. All the recoveries at 5 and 50 µg kg^‐1 (n = 5) ranged between 70% and 120% with RSD below 20%, indicating satisfactory precision. The LOQ for the enantiomers ranged between 0.05 and 1 µg kg^‐1. Based on the proposed method, 135 commonly consumed fruits and vegetables taken from markets in Guizhou province, China, were analyzed. Enantioselective degradation for the selected chiral pesticides was observed in most of the positive samples. Chirality 27:958–964, 2015. © 2015 Wiley Periodicals, Inc.     

Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer

The success of a shotgun proteomic experiment relies heavily on the performance and optimization of both the LC and the MS systems. Despite this, little consideration has, so far, been given to the importance of evaluating and optimizing the MS instrument settings during data‐dependent acquisition mode. Moreover, during data‐dependent acquisition, the users have to decide and choose among various MS parameters and settings, making a successful analysis even more challenging. We have systematically investigated and evaluated the effect of enabling and disabling the preview mode for FTMS scan, the number of microscans per MS/MS scan, the number of MS/MS events, the maximum ion injection time for MS/MS, and the automatic gain control target value for MS and MS/MS events on protein and peptide identification rates on an LTQ‐Orbitrap using the Saccharomyces cerevisiae proteome. Our investigations aimed to assess the significance of each MS parameter to improve proteome analysis and coverage. We observed that higher identification rates were obtained at lower ion injection times i.e. 50–150 ms, by performing one microscan and 12–15 MS/MS events. In terms of ion population, optimal automatic gain control target values were at 5×10⁵–1×10⁶ ions for MS and 3×10³–1×10⁴ ions for MS/MS. The preview mode scan had a minimal effect on identification rates. Using optimized MS settings, we identified 1038 (±2.3%) protein groups with a minimum of two peptide identifications and an estimated false discovery rate of ～1% at both peptide and protein level in a 160‐min LC‐MS/MS analysis.