UMass Boston

Announcements

• The Proteomics Core Facility is excited to announce that they have been awarded a MLSC grant to fund a new Bruker timsTOF HT. Stay tuned, coming Spring 2023! 

• The Proteomics Core Facility is excited to announce the implementation of iLab! 

The University of Massachusetts Boston Proteomics Core offers a full range of proteomic services using the most advanced instrumentation and methodologies. We offer unmatched customer support based on extensive experience in biological mass spectrometry. Our services range from sample preparation, bottom-up protein identification, quantification of expression levels using both label free and TMT multiplexing strategies, PTM analysis, 2D fractionation, and top-down proteomic analysis. Our instrumentation offers unique flexibility in designing proteomic experiments to provide the best outcome for your project. The director of the core, Professor Jason Evans, has over 25 years of experience in the field of mass spectrometry and proteomics, and as a facility we have been servicing the proteomics needs of multiple principle investigators at the University of Massachusetts Boston since the Spring of 2016. We take the time to consult with you to learn your needs and formulate the best plan for analyzing your samples, so that you get the most out of the resulting data. We work very hard to keep our turn-around times as short as possible. In addition, as a core facility at the University of Massachusetts Boston, we are currently in a unique position to offer our services to small businesses with less than 10 employees at a 75% discount and to small businesses with less than 50 employees a 50% discount from subsidies provided though the state of Massachusetts Innovation Voucher Program.

Table of Contents

• Services and Pricing
• Getting Started with iLab
• Equipment
• What A Typical Process Looks Like
• Useful Definitions
• Contact Us

Services and Pricing

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Extraction and Digestion 

The Proteomics Core will extract and digest your proteins of interest using the Pierce Mass Spec Sample Prep Kit for Cultured Cells protocol unless otherwise discussed. 

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1D-LC-MS (90 min. Gradient) 

Typically, this consists of the sample(s) being run using the Easy-nLC 1200 with a 90-minute data-dependent CID/ETD decision tree method. However, during consultation with the core, other gradients and methods can be discussed. The raw data is then processed using Thermo Proteome Discoverer. 

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2D-LC-MS 

This service is appropriate for whole cell tryptic digests. The sample is fractionated on the UltiMate 3000 RS system using high pH reversed phase HPLC into ten fractions and each fraction is analyzed using the 90 minute decision tree LC-MS method. The raw data will be processed with Thermo Proteome Discoverer. Using this method, the core identifies over 12,000 proteins in a HeLa cell digest. The list price includes the total cost of the analysis and not just the first dimensional fractionation. 

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Intact Protein Analysis

The Proteomics Core will analyze purified protein samples via top-down LC/MS, using an HPLC column ideal for proteins ranging from 10-150 kDa via high resolution ETD/HCD mass spectrometry. The data will provide you with molecular weight confirmation and we will be able to assess the presence of protein variants and attempt to locate PTM sites. 

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TMT Labeling

The Proteomics Core will label your samples with TMT duplex, 6plex, 10plex, or 16plex reagents depending on your multiplexing strategies. The samples will then be combined and run as a single sample using the MS³ capabilities of the Orbitrap Fusion Lumos Tribrid. The tryptic peptides will be fragmented using CID (MS² spectra) and quantified using HCD (MS³ spectra). Quantification data is reported in the Proteome Discoverer result file as abundance values. These abundance values are determined for the reporter ion of each PSM (peptide spectrum match) via identification of the minimum detected quantification value per spectrum file. Peptide abundances are then determined via a simple summation of its associated and used PSM abundances. Protein abundances are found in a similar manner, using the simple summation its associated and used peptide abundances. 

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Label Free Quantification

The Proteomics Core will provide processed data complete with area values as determined by Proteome Discoverer. Proteome Discoverer determines these area values by determining the maximum PSM (peptide spectrum match) area under the curve of the extracted ion chromatogram. It then sets this area as the area of the peptide group to which the PSM belongs. Protein areas are calculated as an average of the top 5 peptide group areas associated with that protein. 

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Small Molecule Exact Mass/CID

The Proteomics Core can provide exact mass and fragmentation data for purified small molecule applications. Download the HRMS Submission Form to provide the core with detailed information about your small molecule(s). 

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Complete mAb Characterization

The Proteomics Core can provide a complete characterization of antibodies. This includes: intact exact mass, intact deglycosylated mass, subunit analysis, deglycosylated subunit analysis, and glycopeptide analysis complete with peptide sequencing and glycoform maps. 

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Getting Started with iLab

The Proteomics Core is excited to start using iLab, an online system to streamline the process of billing for core services along with the Biophysical Instrumentation Core (BIC) Facility and the Scanning Electron Microscope (SEM). All facility users are invited to use the system, which requires a one-time registration as discussed below. Once you are registered, the system will enable you to provide required approvals, input your fund or PO number, complete and submit a sample submission form and monitor the progress of your submission.

To Register for an Account:

• Complete the registration form on the sign-up page.
• Receive a Welcome Email from iLab (typically within one business day) with log-in credentials.
• Log in to our Chemistry Cores page here.
• In the upper-right-hand corner click ‘Sign In’ A pop-up window should appear displaying, ‘Sign in using iLab credentials.’ Click the iLab link. 
• Enter the credentials received in your welcome email from iLab.
• A member of the Proteomics Core staff will input the agreed upon services. Once added, the client will be able to review the services and approve them.
• When ready to submit samples, select the Request Services tab. Here you can request the sample submission service or view our price list.
• To start the sample submission request, click on the ‘Initiate Service’ button next to the Sample Submission (Proteomics) service.
• You will be asked to complete a form and provide payment information for your request before submitting the request to the core.
• Your request will be pending review by the core. The core will add charges and submit it back to you for approval. Make sure to watch for an email from iLab regarding your updated project.

Instructions for PIs

If you have researchers who use the core’s services you may receive email requests from researchers wishing to join your group. The request email will have specific instructions on how to approve the request. In case you are interested in the process, we have pasted instructions below.  If you would prefer to delegate these notifications/approvals to a financial manager, please email iLab Support with your financial manager’s name & email.

Instructions: 

• Click here to log in
• You will use your iLab credentials to login.
• Once logged in, look for the link in the left hand menu that says 'my groups'. Hover-over and select your lab.
• Set the auto-approval amount if you do not wish to approve service requests below a certain dollar amount. To do this, select the 'Members' panel and enter a dollar amount in the 'Auto Pre-Approval' amount and click 'save settings.’
• To approve lab membership requests, select the ‘Membership Requests’ tab. New membership requests will show at the top of this page. Click “Approve” to accept a member into your lab. Click “Reject” if they are not a member of your lab.

Additional Help

More detailed instructions are available on the iLab helpsite. For any questions not addressed, click on the “HELP” link in the upper right hand corner or contact iLab Support.

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Equipment 

The Proteomics Core currently uses the state-of-the-art Orbitrap Fusion Lumos Tribird mass spectrometer with the Easy-ETD upgrade coupled with either an Easy-nLC 1200 or an UltiMate 3000RS HPLC. The core uses the Thermo Xcalibur suite of programs to run the equipment and analyze the raw data files. For bottom-up studies the core uses Thermo Proteome Discoverer to identify and/or quantify the expressed proteins in the client's sample(s). For top-down studies the core uses the ProSightLite application.

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What A Typical Process Looks Like

The Proteomics Core works hard to ensure quality results and expedient turn around times. To this end, and in an effort to be transparent, we have shared below what we envision a typical process encompasses from start to finish. We would like to note that every experiment/investigation is unique and thus clients should expect their individual processes in working with the core to be tailored to suite their specific needs. However, most will following this general sequence of events:

• An initial consultation with the core, either in person or over the phone. This consultation serves to introduce the client to the core and the core to the client. During this initial meeting the general scope of the client's project will be discussed as well as an anticipated strategy in analyzing the samples to ensure the highest quality of data is achieved in a timely manner.
• Following-up. It may be necessary for subsequent conversations between the client and the core to finalize details before moving forward. 
• Signing the Proteomics Core Research Services Agreement. Typically, the core will send this form with everything filled out except for the payment account and the SPONSOR box which requires the client's name, title, email, and electronic signature (which can be done in Adobe Acrobat following this tutorial). This document summarizes the work to be done by the core, the cost of the work, and how the funds will be exchanged. 
• Getting the samples to the core. This can either be an in-person drop-off or the samples can be sent to us through the mail.
• Running the samples. The core will run the samples as per the Research Services Agreement. Communication between the core and the client is paramount and thus any problems or necessary modifications to the agreed upon Research Services Agreement will be appropriately discussed. If a large number of samples were submitted to the core for analysis, periodic updates from the core may be sent. 
• Processing the raw data. If desired, the core will process the raw data using either Proteome Discoverer (bottom-up proteomics) or ProSight Lite (top-down proteomics). 
• Returning the data to the client. Once data collection and processing are completed the core will work with the client to ensure their data is securely returned to them. This can be done either with an in-person visit to campus or electronically (via email or uploading to the cloud). The core can return processed data as either a Proteome Discoverer (.pdResult) file if the client has access to Proteome Discoverer or as an Excel spreadsheet. See the Useful Definitions page for help interpreting processed data. 
• Exit consultation. Once the work is complete and the client has had some time to review the data, there will be at least one more consultation between the client and the core. This goal of this meeting will be to discuss the data, assess the overall satisfaction of the client, and address any questions or concerns that the client might have. 

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Useful Definitions

Understanding the output of Proteome Discoverer (PD) can be aided with these useful definitions. Navigate this page using the links below or using Ctrl-F on Windows (or Cmd-F on a Mac). 

Source: Proteome Discoverer 2.1.0.81 Help Contents. 

• Proteins
• Peptide Groups
• PSMs

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Proteins

Protein FDR Confidence

The level of confidence for the identified protein as determined by the false discovery rate (FDR). In the most common workflows used by the core high confidence (green) has a FDR of 1%, medium confidence (yellow) has an FDR of 5%, and low (red) has a FDR of 10%. This can be interpreted as high confidence hits being 99% accurate, medium being 95% accurate, and low being 90% accurate. 

Master

Denotes if the protein is the master protein of a protein group. 

Accession

The unique identifier for the identified protein by the FASTA database used. The core will use UniProt and Swiss-Prot interchangeably. 

Description

The name of the protein associated with the Accession identifier. 

Exp. q-value

The experimental q-values that are derived from the validation. These are calculated from the number of target and decoy proteins and are the minimum false discovery rate required for a hit to be considered correct. For our common workflows, q-values are greater than 0.01 for high confidence hits and 0.05 for medium confidence hits. 

Sum PEP Score

This score is calculated on the basis of the posterior error probability (PEP) values of the (peptide spectrum matches) PSMs. The PEP is the probability that the observed PSM is incorrect.

Coverage

The percentage of the protein that is covered by the identified peptides.

#PSMs

The number of peptide spectrum matches (PSMs) found for all of the peptides (including redundant) of the identified protein.

Areas (if applicable)

The summation of the areas under the extracted ion chromatograms per PSM per peptide identified for the identified protein. 

Abundances (if applicable) 

With TMT labeled samples, abundance values indicate the amount of protein found in the sample per TMT Reporter Ion. Proteome Discoverer determines abundance values at the PSM (peptide spectrum match) level. These are summed for peptide abundances. The abundances of the peptides are then summed to give the abundances at the protein level. 

emPAI 

The exponentially modified protein abundance index (emPAI) is a simple measurement of protein abundance based on the number of identified peptides for that protein. It correlates to the absolute amount of protein in a sample. 

Score SEQUEST HT

The protein score which is calculated by summing the individual scores of each peptide. The higher this score, the higher the individual scores of the peptides, and thus the better the identification. SEQUEST HT is the name of the employed search engine. 

# Peptides SEQUEST HT

The number of distinct peptide sequences in the identified protein. SEQUEST HT is the name of the employed search engine. 

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Peptide Groups

Confidence

The confidence level associated with the identification of the peptide group. A green circle indicates high confidence, a yellow circle indicated medium confidence, and a red circle indicates low confidence. 

Qvality PEP 

This score is calculated on the basis of the (posterior error probability) PEP values of the PSMs (peptide spectrum matches). The PEP is the probability that the observed PSM is incorrect. Qvality is the search node employed. 

Qvality q-value

The experimental q-values that are derived from the validation. These are calculated from the number of target and decoy proteins and are the minimum false discovery rate required for a hit to be considered correct. For our common workflows, q-values are greater than 0.01 for high confidence hits and 0.05 for medium confidence hits. 

Areas (if applicable)

The summation of the areas under the extracted ion chromatograms per PSM per peptide identified. 

Abundances (if applicable) 

With TMT labeled samples, abundance values indicate the amount of protein found in the sample per TMT Reporter Ion. Proteome Discoverer determines abundance values at the PSM (peptide spectrum match) level. These are summed for peptide abundances.  

XCorr SEQUEST HT

The score that indicates the number of fragment ions that are common to two different peptides with the same precursor mass and calculates the cross-correlation (XCorr) for all candidate peptides in search. In other words, the XCorr score measures the fit of the experimental peptide fragments to the theoretical spectra. In general, an XCorr value greater than 2 is considered favorable. Any identification with an XCorr less than 2 should analyzed further to determine goodness of fit. 

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PSMs (Peptide Spectrum Matches)

Confidence

The confidence level associated with the identification of the peptide sequence. A green circle indicates high confidence, a yellow circle indicated medium confidence, and a red circle indicates low confidence. 

PSM Ambiguity

The grouping status of the PSM. Options include:

• Unambiguous: This PSM is the only match and there is no ambiguity
• Selected: This PSM was selected from a group of many matches that it considered for the protein group interference process. 
• Rejected: This PSM was rejected from a group of many matches that were considered. 
• Ambiguous: Two or more PSMs were considered for the same spectrum and could not be distinguished. 
• Unconsidered: The PSM was not considered to be a match due to protein group interference. 

deltaScore

A measure of the difference between the top scores for the peptides identified by the particular spectrum.

deltaCn

The normalized score difference between the selected PSM and highest-scoring PSM for the spectrum. . 

Percolator q-value

The experimental q-values that are derived from the validation. These are calculated from the number of target and decoy proteins and are the minimum false discovery rate required for a hit to be considered correct. For our common workflows, q-values are greater than 0.01 for high confidence hits and 0.05 for medium confidence hits. 

Peculator PEP 

This score is calculated on the basis of the posterior error probability (PEP) values of the PSMs (peptide spectrum matches). The PEP is the probability that the observed PSM is incorrect. 

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Contact Us

Core Director 

Jason Evans, PhD
University of Massachusetts Boston
Office: ISC 03-3410
100 Morrissey Blvd
Boston, MA 02125
Telephone: 617.287.6149
Email: Proteomics.Core@6lwboc.com 

Location of Core

Integrated Sciences Complex, 3rd Floor, Rooms 3341 & 3640

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