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Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.

The Mascot score (M-score) is one of the conventional validity measures in data base identification of peptides and proteins by MS/MS data. Although tremendously useful, M-score has a number of limitations. For the same MS/MS data, M-score may change if the protein data base is expanded. A low M-value may not necessarily mean poor match but rather poor MS/MS quality. In addition M-score does not fully utilize the advantage of combined use of complementary fragmentation techniques collisionally activated dissociation (CAD) and electron capture dissociation (ECD). To address these issues, a new data base-independent scoring method (S-score) was designed that is based on the maximum length of the peptide sequence tag provided by the combined CAD and ECD data. The quality of MS/MS spectra assessed by S-score allows poor data (39% of all MS/MS spectra) to be filtered out before the data base search, speeding up the data analysis and eliminating a major source of false positive identifications. Spectra with below threshold M-scores (poor matches) but high S-scores are validated. Spectra with zero M-score (no data base match) but high S-score are classified as belonging to modified sequences. As an extension of S-score, an extremely reliable sequence tag was developed based on complementary fragments simultaneously appearing in CAD and ECD spectra. Comparison of this tag with the data base-derived sequence gives the most reliable peptide identification validation to date. The combined use of M- and S-scoring provides positive sequence identification from >25% of all MS/MS data, a 40% improvement over traditional M-scoring performed on the same Fourier transform MS instrumentation. The number of proteins reliably identified from Escherichia coli cell lysate hereby increased by 29% compared with the traditional M-score approach. Finally S-scoring provides a quantitative measure of the quality of fragmentation techniques such as the minimum abundance of the precursor ion, the MS/MS of which gives the threshold S-score value of 2.

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.

A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.

The complexity of the uman proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at ≥1 % level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.

Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano-LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, α_s1- casein from human milk was selected as a model molecule representing moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other minerals to the new-born. Human α_s1-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found that the sequence region between the residues Ser70 and Ser76 in human α_s1-casein is in fact phosphorylated, contrary to previous knowledge. The site of the most abundant phosphorylation is Ser75, in agreement with the known action of the mammary gland casein kinase. There is evidence for the second phosphorylation in that region, possibly at Ser73. Earlier reported positions of phosphorylations at Ser18 and Ser26 are also confirmed, but not the dominance of Ser18 phosphorylation. The occupancy rates at Ser18, Ser26 and Ser75 are estimated to be (7 ± 2), (20 ± 6) and (27 ± 9)%, respectively. Owing to differences in the ionization efficiency between phosphorylated and unphosphorylated peptides a 30% error margin is added to the occupancy rates. The highlighted pitfalls of the bottom-up strategy include the sensitivity of enzymes to proximal acidic and phosphorylated residues and the presence of multiple isoforms, including unexpected ones, of the tryptic peptides. The utility of the earlier introduced PhosTS_hunter and ModifiComb approaches for evading the latter pitfall is demonstrated.

(Chemical Equation Presented) Complementary cleavage: A large-scale (ca. 15000 spectra) comparison between the cleavage sites and the preferences of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) in tandem mass spectrometry has proved beyond doubt their complementary nature (see picture, red: high, blue: low). The study has also suggested the presence of a preferred N-terminal structure in gas-phase tryptic peptide dications.

Hydrogen rearrangement is an important process in radical chemistry. A high degree of H· rearrangement to and from z· ionic fragments (combined occurrence frequency 47% compared with that of z·) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron capture dissociation (ECD), including previously unreported double H· losses. Consistent with the radical character of H· abstraction, the residue determining the formation rate of z′ = z· + H· species is found to be the N-terminal residue in z· species. The size of the complementary c_m′ fragment turned out to be another important factor, with z′ species dominating over z· ions for m ≤ 6. The H· atom was found to be abstracted from the side chains as well as from α-carbon groups of residues composing the c′ species, with Gln and His in the c′ fragment promoting H· donation and Asp and Ala opposing it. Ab initio calculations of formation energies of ·A radicals (A is an amino acid) confirmed that the main driving force for H· abstraction by z· is the process exothermicity. No valid correlation was found between the N{single bond}C_α bond strength and the frequency of this bond cleavage, indicating that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing.

Analysis of a database of some 20 000 conventional electron-capture dissociation (ECD) mass spectra of doubly charged ions belonging to tryptic peptides revealed widespread appearance of w ions and related u ions that are due to partial side chain losses from radical z^. ions. Half of all z^. ions that begin with Leu or Ile produce w ions in conventional one-scan ECD mass spectra, which differentiates these isomeric residues with >97% reliability. Other residues exhibiting equally frequent side chain losses are Gln, Glu, Asp, and Met (cysteine was not included in this work). Unexpectedly, Asp lost not a radical group like other amino acids but a molecule CO₂, thus giving rise to a radical w^. ion with the possibility of a radical cascade. Losses from amino acids as distant as seven residues away from the cleavage site were detected. The mechanism of such losses seems to be related to radical migration from the original site at the ^αC_n atom in a Z_n. ion to other ^αC and ^βC atoms. The side chain losses confirm sequence assignment, improve the database matching score, and can be useful in de novo sequencing.

A strong correlation is found between the propensity of individual amino acids to induce peptide-bond cleavage in the gas phase (P_AA-XX) and their structure-forming propensity (P_S, red) and H-bond-accepting propensity (P_H, blue). Thus, the same fundamental parameter, carbonyl group basicity, governs the formation of secondary protein structures in solution and directs fragmentation in the gas phase. (Graph Presented).