AI-64310-CRFT

Targeted Multiplexed Protein Quantitation Using Serial Immunoaffinity Extraction Coupled to LC-MS

Overview

Purpose:

Evaluate the reproducibility of serial protein extraction

from one sample on an individual or multiple protein extraction.

Methods:

Perform HR/AM MS analysis of MSIA D.A.R.T serial

extractions from a single sample using one Ab for a single

protein or multiple Abs (one per D.A.R.T tip).

The relative

quantitation on peptides was used to determine depletion and

disruption effects from serial dilution.

Results:

Pooling of serial extractions increased targeted peptide

response ca. 5-10x without significantly increasing background

interference. The increased abundance resulting from pooling

extractions facilitated greater protein sequence coverage and

detection of N- and O-linked glycopeptides and corresponding

glycoforms.

Introduction

Targeted protein quantitation using LC-MS provides significant

advantages to determining protein sequence variations,

truncations, and/or PTMs. To extend the sensitivity,

immunoaffinity extraction based on antibody (Ab) capture has

been implemented. The benefits of Ab capture for LC-MS

studies increases the targeted protein amounts while

significantly reducing background matrix effects facilitating faster

LC gradients and greater sequence coverage. Performing Ab

extraction using Mass Spectrometry ImmunoAffinity (MSIA)

capture does not disrupt the sample. This facilitates serial

extractions of low sample volumes yet facilitating multiplexed

experiments.

Methods

Sample Preparation

A common stock plasma sample was used for all studies. A

total of 1 µg of digest was prepared and analyze. A 200 µL

aliquot was used for each sample. A set of Thermo Fisher

Scientific™ MSIA™ D.A.R.T. tips were used with each tip

covalently bound to a specific Ab used to extract a set of

targeted proteins. Targeted protein extraction was performed by

serial extraction where a new tip was inserted into the same

sample well and aspirated prior to collection, reduction,

alkylation, and digestion. LC-MS analysis was performed on

each individual extraction or pooled for multiplexing.

The first set of experiments used two different samples for the

analysis of serial extraction. A total of six extractions were

performed with the extracted protein separately collected from

each tip, reduced, alkylated, and digested with trypsin and

analyzed individually. The second where each of the six

extractions were pooled prior to reduction, alkylation, and

digestion followed by LC-MS analysis. (Figure 1) The targeted

proteins for the first set of experiments was serotransferrin and

zinc-

-2-glycoprotein.

A second set of samples were prepared following the same

approach targeting four different proteins: serotransferrin, zinc-

-2-glycoprotein,

-1-antitrypsin, and lactotransferrin. Four

different MSIA D.A.R.T. tips were used with each set covalently

bound to the specific Ab. The same set of samples were used

for individual extraction (one tip per sample) followed by

reduction, alkylation, and digestion then LC-MS. The second set

of samples had each vial processed through serial extraction by

different MSIA tip. For example, one sample was first extracted

by anti-serotransferrin, then anti-lactotransferrin etc. and the

extractions were pooled prior to reducti n, alkylation, and

digested and ultimately LC-MS.

Results

Figure 2. Comparative cov

DDA experiments of (2a) di

extracted digested. Figure

serotransferrin digest cove

Proteome Discoverer. The

to either short peptides (3-

mapped by database searc

Liquid Chromatography (

LC separation was perform

Hypersil Gold™ 100 x 1 m

a binary solvent system co

0.1% formic acid in MeCN.

performed over 15 minutes

equilibration.

Mass Spectrometry

All experiments were perfor

Exactive™ mass spectrome

dependent/dynamic exclusi

scheme. Full scan MS spe

setting of 70k and all HCD p

using 15k.

Data Analysis

Initial unbiased data searchi

Scientific™ Proteome Disco

Pinpoint 1.4 to search N- an

Screening Tool. To total list

unmodified) were used to p

all samples. Qualitative ass

overlap with spectral library

tolerance), and isotopic dist

analyzed by evaluating prec

files not expected to contain

FIGURE 3. Comparative a

result of extraction step v

peptide vs. An N-linked gl

consistent across each pe

extracted. The two peptid

HSTIFENLANK