Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Determination of unique amino acid substitutions in protein variants by peptide mass mapping with FT-ICR MS

Peptide mass mapping plays a central role in the structural characterization of protein variants with single amino acid substitutions. Among the 20 standard amino acids found in living organisms, 18, all but Leu and Ile, differ from each other in molecular mass. The mass differences between amino acids range from 0.0364 to 129.0578 Da. The mass of the mutated peptide or the difference between normal and mutated peptides uniquely determines the type of substitution in some cases, and even pinpoints the position of the mutation when the involved residue is found only once in the peptide. Among 75 pairs of amino acid residues that are exchangeable via a single nucleotide replacement, 53 show specific change in exact mass, while only 25 in nominal mass. On the other hand, precise measurement, at least to the third decimal place, greatly enhances the capacity of the peptide mass mapping strategy for structural characterization. This notion was verified by an analysis of three Hb variants using MALDI-FTICR MS. In addition, the baseline resolution of two 1 kDa peptides with a single amino acid difference, Lys or Gln, which have the smallest (0.0364 Da) difference among residues, was achieved by measurement at a mass resolving power of 342,000. The results indicated that the smallest difference, 0.0040 Da between [Delta29.9742 for Glu-Val] and [Delta29.9782 for Trp-Arg], among all types of amino acid substitutions derived from a single nucleotide replacement can be discriminated at the present performance level. Therefore, FTICR MS is capable of identifying all 53 types of substitutions, each of which is associated with a unique mass difference, except for the Leu and Ile isomers.     

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.     

Peptide structural analysis using continuous Ar cluster and C60 ion beams

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C₆₀ and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn¹ Val⁵]-angiotensin II and [Val⁵]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi₃ ⁺, are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C₆₀ ion beams are useful for peptide structural analysis.     

A method for calculating <Superscript>16</Superscript>o/<Superscript>18</Superscript>o peptide ion ratios for the relative quantification of proteomes

A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using 16O/18O ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/-0.94 ppm (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18O; and the method is not sensitive to variations in 18O1/18O2 ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers.     

Novel Dual Two‐Dimensional Liquid Chromatography Online Coupled to Ultraviolet Detector,Fluorescence Detector,Ion‐Trap Mass Spectrometer for Short Peptide Amino Acid Sequence Determination with Bottom‐Up Strategy

A novel dual two‐dimensional (2D) high‐performance liquid chromatography (LC) setup coupled online to an ultraviolet (UV) detector, fluorescence (FL) detector, and ion‐trap mass spectrometer (MS) has been developed for determining the amino acid sequence of short peptides using a novel bottom‐up strategy. Short peptides were electrothermally hydrolyzed to shorter peptides and amino acid enantiomers. The first 2D LC‐UV and FL system was used to separate and identify the produced parent and daughter short peptides and amino acid isomers and enantiomers in the hydrolysate; the second 2D LC‐MS was used to identify the presence of cysteine and obtain the molecular mass signals and N‐terminal peptide fragment ion signals for parent and daughter short peptides. The identified amino acid enantiomers are used to form any possible short peptides by permutation and combination in an order from dipeptide to a tripeptide, to a tetrapeptide, and to even higher short peptides. The correct short peptides are confirmed by comparing the molecular weights of the constituent amino acid enantiomers and the molecular weights of identified short peptides together, with the characteristic N‐terminal peptide fragment ion signals. The amino acid sequence of the dipeptide ester aspartame and the tripeptide glutathione was successfully determined by this method.     

Dissociation pathways of alkali-cationized peptides: Opportunities for C-terminal peptide sequencing

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.     

ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using <Emphasis Type="Italic">Bacillus subtilis</Emphasis>: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC₅₀ values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Evaluation of temperature,excipients impact on the primary structure of Ganirelix in an injectable formulation,and comparison with Orgalutran® using MALDI‐TOF/TOF‐MS

Ganirelix is a linear polypeptide consisting of covalently bonded 10 amino acid residues. The amino acid sequence in a peptide determines the properties of the molecule. The slightest change in the primary structure (amino acid sequence) of therapeutic peptides can significantly impact its safety, efficacy, and immunogenicity. Hence, the primary structure analysis of therapeutic peptides is regarded as a critical quality attribute (CQA). A vast array of analytical techniques can be used to capture the primary structure of the peptide. In this study, we applied matrix‐assisted laser desorption ionization (MALDI)/tandem time of flight mass spectroscopic (TOF/TOF MS) method to demonstrate the primary structure of Ganirelix in an injectable formulation. The apparent monoisotopic molecular mass of Ganirelix is 1,568.9 Da. The attained primary amino acid sequence of Ganirelix in temperature‐stressed generic product matched with the theoretical sequence and showed homology with those of the reference listed drug (RLD).     

Electron capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

Cooperative effect of factors governing molecular ion yields in desorption/ionization mass spectrometry

Factors governing the molecular ion yields of amino acids and peptides have been studied using fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive-ion mode. The ion yields of protonated amino acids under FAB conditions are dependent on proton affinity (PA), hydrophobicity, and aromaticity of amino acids. Both PA and hydrophobicity contribute to an increase in the ion yields, while aromaticity contributes to a decrease. In MALDI, the ion yields increase linearly with the increase of PA of amino acids with the exception of lysine. In both FAB and MALDI experiments with peptides, the presence of arginine residues is essential for producing abundant protonated peptides. In FAB, the presence of aliphatic and hydrophobic amino acids (leucine and isoleucine) increases the ion yields of protonated peptides, while some hydrophilic amino acids (aspartic acid and asparagines) decrease the ion yields. The presence of two or more arginine residues does not give higher ion yields in FAB. In MALDI, the presence of aromatic amino acids (phenylalanine and tyrosine) enhances the signals for protonated peptides. Thus, physicochemical factors of individual amino acids cooperatively affect the ion yields of protonated amino acids and peptides. These factors governing the ion yields in FAB and MALDI affect two processes, desorption and ionization, that can be considered independently.     

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.     

Fragmentation of cationized phosphotyrosine containing peptides by atmospheric pressure MALDI/Ion trap mass spectrometry

An investigation of phosphate loss from sodium-cationized phosphotyrosine containing peptide ions was conducted using liquid infrared (2.94 microm) atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) coupled to an ion trap mass spectrometer (ITMS). Previous experiments in our laboratory explored the fragmentation patterns of protonated phosphotyrosine containing peptides, which experience a loss of 98 Da under CID conditions in the ITMS. This loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. Phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence. In the absence of a basic residue, the protonated phosphotyrosine peptides do not undergo losses of HPO(3) (Delta 80 Da) nor HPO(3) + H(2)O (Delta 98 Da) in their CID spectra. However, sodium cationized phosphotyrosine containing peptides that do not contain arginine or lysine residues within their sequences do undergo losses of HPO(3) (Delta 80 Da) and HPO(3) + H(2)O (Delta 98 Da) in their CID spectra.     

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.     

Mass spectrometry analysis of synthetically myristoylated peptides

Myristoylpeptides were synthesized in order to determine if a neutral loss of 210 Da, C14H26O (the mass of the myristoyl moiety), was universal and observable by both liquid chromatography electrospray ionization quadrupole ion trap (LC-ESI-QIT) and matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-ToF/ToF) mass spectrometry. Myristoylation was successfully introduced on the N-terminus, internally on the amino group of lysine and arginine. Larger peptides and the arginine compounds needed elevated temperatures for myristoylation. To our knowledge, this is the first report of a chemically-synthesized myristoylated arginine in a peptide. Collision energy studies for the LC-ESI-QIT instrument showed that modified peptides and a loss of 210 Da could be detected under commonly used conditions (energy level between 30 and 42%) with picomole amounts of sample. The loss of myristoyl moiety is observed on the MALDI-Tof/Tof mass spectrometer as well. Due to the hydrophobic properties of the myristoyl moiety, it is not surprising that the modified peptides all formed at least dimers, and in some cases trimers. We were also able to distinguish a mixture of two mono-myristoylated peptides. MS3 data from the LC-ESI-QIT instrument on a di-myristoylated peptide indicates the loss of 210 Da at either the N-terminus or lysine. We were also able to analyze a mixture of modified and unmodified peptides on the MALDI-ToF/ToF instrument. The data presented in this paper demonstrates the constant neutral loss of the 210 Da, C14H26O, from both N-terminally and internally myristoylated peptides can be identified unambiguously using LC-ESI-QIT or MALDI-ToF/ToF mass spectrometers. This will be a useful tool in determining the myristoylation status of candidate proteins after enzyme digestion, and in elucidating the modification sites of internal myristoyl proteins.     

Analysis of native amino acid and peptide enantiomers by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry was used for the separation and detection of amino acid and peptide enantiomers. With detection limits as low as 250 pg, 25 amino acids enantiomers were baseline resolved on a Chirobiotic T chiral stationary phase. APCI demonstrated an order of magnitude better sensitivity over electrospray ionization (ESI) for free amino acids and low molecular mass peptides at the high LC flow-rates necessary for rapid analysis. As the peptide chain length increased (peptides with M(r) > or = 300 Da), however, ESI proved to be the more ideal atmospheric pressure ionization source. A mobile phase consisting of 1% (w/w) ammonium trifluoroacetate in methanol and 0.1% (w/w) formic acid in water increased the sensitivity of the APCI method significantly. A step gradient was then used to separate simultaneously all 19 native protein amino acid enantiomers in less than 20 min using extracted ion chromatograms.     

An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptides

The low‐abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low‐abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time‐of‐flight MS instrument. As a demonstration of the approach, two low‐abundance peptides with masses of ～4000–5000 Da were selected for MS/MS sequencing. The multi‐channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C‐terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery. Copyright © 2009 John Wiley & Sons, Ltd.