MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism, and differentiation. Isobaric labeling, for example, tandem mass tags (TMT), in phosphoproteomics workflows enables both relative and absolute quantitation of these phosphorylation events. Traditional TMT workflows identify peptides using fragment ions at the MS2 level and quantify reporter ions at the MS3 level. However, in addition to the TMT reporter ions, MS3 spectra also include fragment ions that can be used to identify peptides. Here we describe using MS3 spectra for both phosphopeptide identification and quantification, a process that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize several instrument parameters, including the modality of mass analyzer (i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and MS3 normalized collision energy (NCE), to achieve the best balance of identified and quantified peptides. Our optimized MS3-IDQ method included the following parameters for the MS3 scan: NCE = 37.5 and AGC target = 1.5 × 10⁵, and scan range = 100–2000. Data from the MS3 scan were complementary to those of the MS2 scan, and the combination of these scans can increase phosphoproteome coverage by >50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides

Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags

Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons

Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags

Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons

Increasing Throughput in Targeted Proteomics Assays: 54-Plex Quantitation in a Single Mass Spectrometry Run

Targeted proteomics assays such as those measuring end points in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included three biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents, etc.) and a ∼50-fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of nine compounds

Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags

Isolation of SelP from culture media of HepG2 cells.

<p>Medium samples from HepG2 cells were chromatographed on HisPur resin. Fractions were analyzed by SDS-PAGE, the gels stained with Coomassie blue (<i>Lower panels</i>) and subjected to Western blotting with anti-SelP antibodies (<i>Upper panels</i>). <i>Lane 1</i>, initial sample, <i>lane 2</i>, flow-though fraction, <i>lane 3</i>, buffer wash, <i>lane 4–10</i>, elution with a gradient of imidazole from 0 to 200 mM in loading buffer. (A) HepG2 cells grown on DMEM medium only (control). (B) HepG2 cells grown on DMEM supplemented with 100 nM Se. (C) HepG2 cells grown on DMEM supplemented with 1 mM thiophosphate (SPO₃). Protein molecular weights markers in kDa are shown on the <i>left</i>. Experimental details are given in <i>Materials and Methods</i>.</p

Human SelP sequence.

<p>Positions of the ten Sec residues are indicated by numbers and shown in red. Positions of Cys residues are highlighted in bold. Natural His-rich segments, which allow affinity isolation of human SelP, are underlined.</p

SelP expression in hepatocytes and its regulation by selenium and thiophosphate.

<p>(A) HepG2 cells were cultured without or with 100 nM Se or 1 mM thiophosphate (SPO3), metabolically labeled with ⁷⁵Se, and cell lysates and culture media samples analyzed by SDS-PAGE and PhosphorImager for selenoprotein expression. Positions of SelP and GPx3 are indicated by <i>arrows</i> on the right, and protein molecular weight markers are indicated on the left. (B) Samples from panel <i>A</i> were analyzed by Western blotting (WB) with anti-SelP antibodies. Positions of protein molecular weight markers are indicated on the left and SelP is indicated by <i>arrows</i> on the right. (C) Coomassie blue (<i>CBB</i>) staining of samples from <i>A</i> and <i>B</i>, used as a loading control. (D) SelP isolated from human blood was analyzed by SDS-PAGE and stained with Coomassie blue. Protein molecular weights markers in kDa are shown on the <i>left</i>. Positions of different SelP forms are indicated by <i>arrows</i>. Position of serum albumin is indicated by an <i>asterisk</i>.</p

Tryptic Sec-containing peptides of SelP isolated from pooled (n = 5) human plasma.

<p>Percent Sec and Cys in SelP peptides was assessed by MS/MS as described in Materials and Methods.</p><p>Tryptic Sec-containing peptides of SelP isolated from pooled (n = 5) human plasma.</p