Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling

Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to the target species. External calibration was employed as a quantification technique. The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column. The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme and bovine serum albumin.     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

Peptide and protein quantification using iTRAQ with electron transfer dissociation

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c- and z -type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-C(alpha) bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error < 10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.     

PSAQ™ standards for accurate MS–based quantification of proteins: from the concept to biomedical applications

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable‐isotope‐labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ?) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here. Copyright © 2012 John Wiley & Sons, Ltd.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative liquid chromatography tandem mass spectrometry analysis of macromolecules using signature peptides in biological fluids

Targeted protein quantification using peptide surrogates has increasingly become important to the validation of biomarker candidates and development of protein therapeutics. These approaches have been proposed and employed as alternatives to immunoassays in biological fluids. Technological advances over the last 20 years in biochemistry and mass spectrometry have prompted the use of peptides as surrogates to quantify enzyme digested proteins using triple quadrupole mass spectrometers. Multiple sample preparation processes are often incorporated to achieve quantification of target proteins using these signature peptides. This review article focuses on these processes or hyphenated techniques for quantification of proteins with peptide surrogates. The most recent advances and strategies involved with hyphenated techniques are discussed.     

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography‐multiple reaction monitoring mass spectrometry (LC‐MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope‐labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC‐MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Interchain doubly-bridged α-helical peptides for the development of protein binders

This work reported the design and synthesis of interchain doubly-bridged α-helical peptides, involving mutual stabilization of two α -helical peptides crosslinked by two interchain bisthioether crosslinkers.     

Normalization of relative peptide ratios derived from in-gel digests: applications to protein variant analysis at the peptide level

The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80,000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the alpha/beta-spectrin self-association sites were added to purified digested alpha-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.     

Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

Absolute quantification of NAD(P)H:quinone oxidoreductase 1 in human tumor cell lines and tissues by liquid chromatography–mass spectrometry/mass spectrometry using both isotopic and non-isotopic internal standards

NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) is a prognostic biomarker and a potential therapeutic target for various tumors. Therefore, it is of significance to develop a robust method for the absolute quantification of NQO1. This study aimed to develop and validate a LC–MS/MS based method and to test the appropriateness of using non-isotopic analog peptide as the internal standard (IS) by comparing with a stable isotope labeled (SIL) peptide. The chromatographic performance and mass spectra between the selected signature peptide of NQO1 and the non-isotopic peptide were observed to be very similar. The use of the two internal standards was validated appropriate for the absolute quantification of NQO1, as evidenced by satisfactory validation results over a concentration range of 1.62–162 fmol μL⁻¹. This method has been successfully applied to the absolute quantification of NQO1 expression in various tumor cell lines and tissues. NQO1 expression in human tumor tissues is much higher than that in the neighboring normal tissues in both the cases of lung and colon cancer. The quantitative results obtained from the isotopic and non-isotopic methods are quite similar, further supporting that the use of non-isotopic analog peptide as internal standard is appropriate and feasible for the quantification of NQO1. By comparing with a classical isotopic IS, the present study indicates that the use of a non-isotopic peptide analog to the proteotypic peptide as the internal standard can get equal accuracy and preciseness in measuring NQO1. The universal applicability of the non-isotopic IS approach for the quantification of proteins warrants further research.     

Development of an absolute quantification method targeting growth hormone biomarkers using liquid chromatography coupled to isotope dilution mass spectrometry

A method to perform absolute quantification of two biomarkers (IGF-1 and IGFBP-3) of growth hormone abuse has been developed. Isotope dilution is used with synthetically labelled peptides as internal standards. Peptide selection and multiple reaction monitoring design are discussed. A simple sample preparation based on the reduction and alkylation of cysteine residues followed by tryptic digestion provides a sufficient digestion of proteins. Serum samples fortified with increasing amounts of target proteins are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer. Specificity is ensured by the selection of sequences with no homology in BLAST, as well as retention time deviation check, and ion ratio monitoring. Linearity is studied in terms of calibration curves. These curves for IGFBP-3 and IGF-1 are generated with mean slopes of 0.055 and 0.065, intercepts of 0.107 and -0.011, and with coefficients of correlation of 0.95 and 0.98, respectively. These curves result from the addition of proteins to the serum. Risks of variations related to potential matrix effects are therefore reduced, as well as probable variations related to the digestion steps. The working concentration ranges are 4-10 ng/microl for IGFBP-3 and 2-8 ng/microl for IGF-1. Preliminary data regarding repeatability show that relative standard deviations (RSDs) range between 13 and 32% for IGFBP-3 and between 7 and 29% for IGF-1.     

Compensation of gradient related effects when using capillary liquid chromatography and inductively coupled plasma mass spectrometry for the absolute quantification of phosphorylated peptides

The application of reversed phase liquid chromatography (RP-LC) hyphenated to inductively coupled plasma mass spectrometry (ICP-MS) for the accurate quantification of bio-molecules via covalently bound hetero atoms such as phosphorus is restricted, due to the known effects of increasing amounts of organic solvents on the ionization behavior of certain elements. An approach for the compensation of variations in the elemental response, due to changes in the solvent composition during the RP gradient separation of phosphorylated peptides is described, which includes the application of a second, matched reversed gradient, that is mixed post-column with the RP column outflow before entering the LC–ICP-MS interface. The experimental design allows the application of gradient separations, while the element-specific detection is carried out under isocratic conditions with a constant organic solvent intake into the plasma. A constant elemental response is a general pre-requisite for the application of ICP-MS for the absolute quantification of peptides via their hetero atom content, especially when no corresponding high purity standards are available or natural mono-isotopic hetero element tags are utilized. As complementary technique LC–electrospray ionization linear ion trap mass spectrometry (ESI-QTRAP-MS) has been used for peptide identification and to elucidate their phosphorus stoichiometry. Highly reproducible separations have been obtained with retention time and peak area RSDs of 0.05% and 7.6% (n = 6), respectively. Detection limits for phosphorus of 6 μg L⁻¹ (6 pg absolute), have been realized, which corresponds to approximately 200 fmol of an average molecular weight, singly phosphorylated peptide. In addition an automatic routine for flow injection analysis (FIA) at the end of each chromatographic separation has been developed, to calibrate each chromatographic separation, which allows absolute quantification of the separated species, whenever their tag stoichiometry is known. Phosphorylated peptides as well as tryptic protein digests have been used as model compounds for method development and to demonstrate the applicability of the proposed setup for phosphopeptide quantification on the basis of simple inorganic phosphorus standards.     

Absolute quantification of cardiac troponin T by means of liquid chromatography/triple quadrupole tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric method for absolute quantification of cardiac troponin T (cTnT) in mouse heart tissue is presented. Even in such a complex biological sample, the multiple reaction monitoring acquisition mode allowed the selective and sensitive determination of a specific peptide, obtained by cTnT enzymatic digestion. The concentration of this cTnT-specific peptide was considered as a representation of the concentration of its parent protein. Quantification was carried out by means of the matrix-matched calibration curve, constructed by adding the synthetic standard of the target peptide and another synthetic structurally analogous peptide as internal standard. Method identification limit and method quantification limit were estimated as 60 and 110 ng of cTnT per mg of total extracted proteins, respectively. The developed label-free approach has been applied for the absolute quantitation of cTnT because of its diagnostic and prognostic value as cardiac disease marker. However, the method could be of general application, since it requires only the synthesis of two suitable peptides, a protein tryptic cleavage product and an internal standard.     

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Targeted data acquisition for improved reproducibility and robustness of proteomic mass spectrometry assays

Quantitative mass spectrometry-based proteomic assays often suffer from a lack of robustness and reproducibility. We here describe a targeted mass spectrometric data acquisition strategy for affinity enriched subproteomes—in our case the kinome—that enables a substantially improved reproducibility of detection, and improved quantification via isobaric tags. Inclusion mass lists containing m/z, charge state, and retention time were created based on a set of 80 shotgun-type experiments performed under identical experimental conditions. For each target protein, peptides were selected according to their frequency of observation and isobaric tag for relative and absolute quantitation (iTRAQ) reporter ion quality. Retention times of selected peptides were aligned using similarity driven pairwise alignment strategy yielding <1 min standard deviation for 4 h gradients. Multiple fragmentation of the same peptides resulted in better statistics and more precise reporter ion based quantification without any loss in coverage. Overall, 24% more target proteins were quantified using the targeted data acquisition approach, and precision of quantification improved by >1.5-fold. We also show that a combination of higher energy collisional dissociation (HCD) with collisional induced dissociation (CID) outperformed pulsed-Q-dissociation (PQD) on the OrbitrapXL. With the CID/ HCD based targeted data acquisition approach 10% more quantifiable target proteins were identified and a 2-fold increase in quantification precision was achieved. We have observed excellent reproducibility between different instruments, underlining the robustness of the approach.     

Matrix-assisted laser desorption/ionisation mass spectrometric response factors of peptides generated using different proteolytic enzymes

Matrix-assisted laser desorption/ionisation (MALDI) mechanisms and the factors that influence the intensity of the ion signal in the mass spectrum remain imperfectly understood. In proteomics, it is often necessary to maximise the peptide response in the mass spectrum, especially for low abundant proteins or for proteolytic peptides of particular significance. We set out to determine which of the common proteolytic enzymes give rise to peptides with the best response factors under MALDI conditions. Standard proteins were enzymatically digested using four common proteases. We assessed relative response factors by coanalyzing the resulting digests. Thus, when tryptic peptides were added in equimolar quantities to their corresponding Asp-N, chymotrypsin and Glu-C digests, tryptic peptide signals were always predominant in the resulting MALDI mass spectra. Observable peaks attributable to non-tryptic peptides generally contained a terminal basic residue. It was proposed that a terminal basic residue has a disproportionate influence upon gas-phase basicity, and this hypothesis was supported by experiments with model isotopically labelled peptides. Experiments applying Cook's kinetic method showed that the peptide with a C-terminal arginine residue was more basic than the equivalent peptide with an N-terminal arginine, which was more basic than the peptide in which the arginine was mid-chain. Thus, the observation of the higher MALDI mass spectrometry response factors of tryptic peptides in comparison with peptides derived using other proteolytic enzymes corresponds with higher gas-phase basicities and may, along with other factors such as the complexity of the digest, influence the choice of enzyme in "bottom-up" proteomic experiments.